Composition comprising prostacyclin andor analogues thereof for treatment of acute critically ill patients

ABSTRACT

Use of prostacyclin or an analogue thereof for treatment of a new medical indication in acute critically ill patients, in particular acute critically ill patients with systemic endothelial damage, a biomarker for identifying individuals that have a new medical indication, and a method for identifying a new medical indication.

FIELD

The aspects of the disclosed embodiments relate to novel uses ofprostacyclin and analogues thereof for treatment of patients sufferingfrom acute critical illness. The aspects of the disclosed embodimentsalso relate to a method of identifying patients with a newly identifieddisease entity in order to initiate treatment earliest possible,pre-hospital as well as in-hospital, after the injurious hit.

BACKGROUND

Acute critical illness such as trauma, sepsis and resuscitated cardiacarrest affects millions of people worldwide annually with a projected40% increase in global deaths due to injuries in the period 2002 to2030. Approximately one quarter of acute critically ill patients developsevere hemostatic aberrations resulting in impaired clotting ability(coagulopathy); such acute critically ill patients with coagulopathyhaving 3-4 times higher mortality rates compared to theirnon-coagulopathic counterparts (40-50% vs. 10-15%). Importantly, besidesearly mortality related to exsanguination, acute critically ill patientswith coagulopathy have a several-fold increased risk of developing anddying from multiple organ failure in the days and weeks that follow theinjurious hit. Regrettably, the outcome for acute critically illpatients with coagulopathy has remained unaltered since the 1970ies,despite a general improvement in intensive care capabilities, pointingtowards a lack of identification of the pathophysiologic mechanism(s)responsible for the poor outcome [Artenstein et al 2013; Marshall 2014].Currently, no drugs/pharmacological agents or therapeutic interventionsare registered to specifically treat coagulopathy in acute criticallyill patients.

Microcirculatory failure is a hallmark of acute critical illness that iscaused by numerous injurious hits to the vascular system, including theendothelium, i.e. the single layer of cells that lines the interior ofall blood vessels in the body. Although there is emerging consensus thatendothelial damage is a critical contributing factor to the developmentof organ failure and poor outcome in acute critically ill patients [Opaland van der Poll 2015], no drugs or therapies are currently registeredto specifically treat endothelial damage in acute critically illpatients. There is thus an urgent unmet need for diagnostic tests andtherapeutic interventions capable of reversing and treating thedeleterious changes observed in the vascular system, including theendothelium, in acute critically ill patients [Marshall 2014; Simmonsand Pittet 2015].

The Endothelium

The endothelium is the collective thin layer of cells (endothelialcells) that lines the interior of all blood and lymphatic vesselsthroughout the body. It is one of the largest “organs” in the human bodyhaving a total weight of approximately 1 kg and covering a total surfacearea of approximately 4-7,000 m². The luminal surface of the endothelialcells is covered by a 0.2-1.0 μm thick negatively chargedcarbohydrate-rich surface layer, the endothelial glycocalyx, that alsorepresents a large structure in the vascular system by containing afixed non-circulating plasma volume of approximately 1 liter in adults,corresponding to one third of the intravascular plasma volume. Theglycocalyx provides the endothelium with an anti-adhesive andanticoagulant surface that protects the endothelial cells and maintainsvascular barrier function. The glycocalyx is a mesh-like structurecomprising proteoglycan and glycoprotein backbone molecules that bindand incorporate various soluble molecules derived from the plasma andendothelium, with the highest amounts of plasma derived constituentstowards the luminal surface.

The endothelium is critically involved in maintaining the delicatehomeostasis between the circulating blood and all vital organs. Bytraversing each and every organ in the body, the endothelium is pivotalfor maintaining homeostasis between the circulating blood and all vitalorgans and cells of the body and, hence, damage to the endothelium is akey factor of the observed poor outcome in acute critically illpatients. The endothelium controls vasomotor balance, vascularintegrity, blood cell adhesion and trafficking, immune surveillance,inflammation and angiogenesis, and it is instrumental for balancinghemostasis through its release, expression and support of systems andelements that either promote or inhibit hemostasis.

In a healthy state, the endothelium is anticoagulated by constituents ofthe glycocalyx and the endothelial cells themselves. Upon endothelialdamage, these constituents are released to the flowing blood whileretaining their anticoagulant effects, thereby contributing tocoagulopathy of acute critical illness. At the same time, the damagedendothelium becomes prothrombotic and triggers formation ofmicrovascular thrombosis resulting in impaired oxygen delivery totissues, organ failure and ultimately death [Johansson and Ostrowski2010]. Furthermore, endothelial damage disrupts the inter-cellular tightjunctions responsible for maintaining endothelial barrier functionbetween the flowing blood and the tissues. This results in capillaryleakage, which further drives hypotension and oxygen deprivation andthereby contribute directly to multiple organ failure and death [Opaland van der Poll 2015].

Until now, most research on acute critically ill patients withcoagulopathy has been limited to studying circulating factors of singlepathways such as the coagulation-, complement- and inflammatory systemsreadily measured in the plasma of patients. However, recognizing thatthe circulating plasma is only one part of the complex vascular system,which includes blood cells (platelets, leukocytes, red blood cells),microparticles and all the vessels that contain the blood, the inventorshypothesized that the observed plasma aberrations reflect a universal,evolutionary developed response to acute critical illness that isassociated with concurrent changes in the vascular endothelium andcirculating blood cells [Johansson and Ostrowski 2010].

Prostacyclin

Prostacyclin is a naturally occurring prostaglandin released by healthyendothelial cells. In humans, prostacyclin generation by the vascularendothelium is approximately 0.08-0.10 ng/kg/min with a maximalconcentration of 3.4 pg/l in the circulation [Davies and Hagen 1993].Prostacyclin performs its function through a paracrine signaling cascadethat involves G protein-coupled receptors (GPCR) on nearby endothelialcells and platelets.

The two main pharmacologic actions of prostacyclin and its analogues arevasodilation and inhibition of platelet aggregation, which is reflectedby the current medical indications for prostacyclin analogs [includingbut not limited to Iloprost, Epoprostenol, Epoprostenol Sodium,treprostenil sodium, selexipag, Beraprost, etc. administered eitherintravenously (i.v.), subcutaneously (s.c.) or oral (p.o.)]:

-   1) primary pulmonary hypertension (NYHA class III-IV patients)-   2) secondary pulmonary hypertension (NYHA class III-IV patients with    the scleroderma spectrum of disease who do not respond adequately to    conventional therapy)-   3) anticoagulation during hemodialysis or renal replacement therapy    when heparin is contraindicated-   4) peripheral arterial disease with imminent risk of amputation when    surgical treatment and angioplasty is not possible-   5) progressive thrombangitis obliterans (mb. Burger) with critical    limb ischemia when surgical treatment and angioplasty is not    possible

A study in human volunteers reported that prostacyclin at doses lessthan 8 ng/kg/min had no significant effect on systolic or diastolicblood pressure whereas a dose of 8 ng/kg/min reduced diastolic bloodpressure. The study found no effect on systolic blood pressure in dosesup to 16 ng/kg/min [O'Grady et al 1980]. However, several studies havereported that prostacyclin in doses from 5-10 ng/kg/min lowers systolicblood pressure dose-dependently in acute critically ill patients [Bihariet al 1987; Radermacher et al 1995].

In healthy volunteers, 1-4 ng/kg/min prostacyclin infusion did notinfluence blood pressure and in patients suffering from traumatic braininjury [Grande et al 2000; Naredi et al 2001], acute myocardialinfarction treated by percutaneous coronary intervention (PCI) [Holmvanget al 2012], septic shock [Kiefer et al 2001; Lehmann et al 2000] orCABG surgery [Morgera et al 2002], 0.5-2 ng/kg/min prostacyclin infusiondid not negatively influence blood pressure.

In conclusion it was found that prostacyclin dilates all vascular bedsdose-dependently but the hemodynamic effects of low-dose prostacyclininfusion (up to 4 ng/kg/min) are negligible in healthy volunteers and inacute critically ill patients.

Inhibition of Platelet Aggregation

Prostacyclin inhibition of platelet aggregation is mediated throughplatelet expressed GPCR (IP), which upon prostacyclin binding signalsadenylyl cyclase to produce cAMP, which activates PKA to decrease freeintracellular calcium concentrations. The rise in cAMP directly inhibitsplatelet activation (secretion and aggregation) and counteractsincreases in cytosolic calcium resulting from platelet activation byplatelet agonists.

Historically, prostacyclin has been considered to be the most potentendogenous inhibitor of platelet aggregation in the human organism[Moncada et al 1976; O'Grady et al 1980]. However, as for thevasodilatory effect, the antiaggretory effect is highly dose-dependent[Moncada et al 1976; O'Grady et al 1980]. Of paramount importance forthe suggested intervention, the inventors have investigated theanti-thrombotic potential of prostacyclin with functional whole bloodhemostatic assays proven to correlate with clinical bleeding conditionsand transfusion requirements (thrombelastography (TEG) and impedanceaggregometry (Multiplate)) and surprisingly they discovered thatlow-dose prostacyclin infusion had no measurable anti-thromboticeffects.

In addition to the dose-dependent pharmacologic actions of prostacyclin(vasodilation and inhibition of platelet aggregation), there is emergingevidence that endogenously released prostacyclin has a paracrinecytoprotective function that is mimicked by prostacyclin analogs. Thisnotion is considered important for the suggested intervention as acutecritical illness disrupts normal prostacyclin release by endothelialcells leaving the patients with a malfunctioning systemically disruptedvascular endothelium.

Cytoprotection

The cytoprotective action of prostacyclin is mediated throughprostacyclin IP receptors expressed on a broad range of cells includingendothelial cells.

At the endothelial level, prostacyclin cytoprotection results inpreservation and/or promotion of endothelial integrity and endothelialquiescence favoring an anticoagulant, antiadhesive, antiapoptotic andantiinflammatory phenotype of the endothelium. Prostacyclin directlypromotes recruitment of endothelial progenitor cells, which enhancesendothelial re-endothelilization of injured endothelium, inhibitsendothelial apoptosis and prevents mitochondrial uncoupling ofphosphorylation from oxidation in the respiratory chain in conditionswith cellular stress, whereby mitochondrial structure and function ispreserved and apoptosis reduced. Also, prostacyclin improves endothelialintegrity and vascular barrier function by upregulating vascularendothelial (VE)-cadherin, which is responsible for maintaining tightjunctions between endothelial cells. Finally, prostacyclin promotesendothelial quiescence by enhancing endothelial expression of other(besides prostacyclin) natural anticoagulant pathways which all, to somedegree, exerts cytoprotive functions.

In addition to the direct action of prostacyclin on endothelial cells,the influence of prostacyclin on other cells and tissues indirectlyprotect the endothelium. Thus, prostacyclin induced vasodilationmediated through vascular smooth muscle relaxation protects theendothelium by ensuring microvascular perfusion and oxygen supply to(potentially hypoxic) cells and tissues. Also, prostacyclin stabilizeslysozomal and cell membranes in immunologic cells which reducesinflammation hereby preventing bystander activation and potential damageof the endothelium [Zardi et al 2005; Zardi et al 2007]. Finally,prostacyclin induced inhibition of platelet aggregation indirectlypreserves endothelial quiescence as activated platelets promoteendothelial activation.

In conclusion, prostacyclin exerts widespread cytoprotective actionsthat result in preservation and/or promotion of endothelial integrityand quiescence favoring an anticoagulant, antiadhesive, antiapoptoticand antiinflammatory phenotype of the endothelium thus counteracting thepathologic state of the endothelium in systemic endotheliopathicsyndrome.

Definitions

The term “acute critical illness”, is meant to include any conditionrendering the patient in immediate need for intensive care therapy. Thecondition may be caused by any acute and extensive injurious hit to thebody including but not limited to physical trauma, burn injury trauma,infection (hereunder sepsis, severe sepsis, septic shock), systemicinflammatory response syndrome (SIRS), acute myocardial infarction orother thromboembolic events.

The term “intensive care therapy”, also term “organ supportive care”here, may include but is not limited to ventilation therapy (e.g.mechanical ventilation), hemodialysis, vasopressor therapy, fluidtherapy, blood transfusion therapy with administration of red blood cellconcentrates, fresh frozen plasma, platelet concentrates, whole blood orcoagulation factor concentrates, systemic antibiotic and/or antiviraland/or antifungal and/or antiprotozoic therapy, parenteral nutrition,granulocyte infusion, T cell infusion, stem cell infusion, anticoagulantand/or antithrombotic therapy including low molecular weight heparins,administration of corticosteroids, tight glycemic control etc.

The term “trauma” as used herein means any shock or body wound producedby a sudden physical injury such as accident, injury or impact to livingtissue caused by an extrinsic agent such as blast trauma, blunt trauma,penetrating trauma, trauma caused by chemical injury (spills, warfare orintoxication), radiation or burns.

The term “shock” is used in the conventional clinical meaning, i.e.shock is a medical emergency in which the organs and tissues of the bodyare not receiving an adequate flow of blood. This deprives the organsand tissues of oxygen (carried in the blood) and allows the build-up ofwaste products. Shock is caused by five major categories of problems:cardiogenic (meaning problems associated with the heart's functioning);hypovolemic/hemorrhagic (meaning that the total volume of bloodavailable to circulate is low); neurogenic (caused by severe injury tothe central nervous system), septic (caused by overwhelming infection,usually by bacteria) or anaphylactic/allergic (caused by systemichistamine release from immune cells and excessive vasodilation).

The term “treatment with prostacyclin or analogues thereof”, as used inthis application, means intravenous treatment of acute critically illpatients or continued critically ill patients with prostacyclin oranalogues thereof.

The terms “treatment” and “treating” as used herein refer to themanagement and care of a patient for the purpose of combating an acutecondition, disease or disorder. The term is intended to include the fullspectrum of treatments for a given condition from which the patient issuffering, such as administration of prostacyclin or analogues thereoffor the purpose of: ameliorating, alleviating or relieving symptoms orcomplications; delaying the progression of the condition, disease ordisorder; curing or eliminating the condition, disease or disorder;and/or reducing the risk of or preventing the condition, disease ordisorder, including preventing recurrence of the disease, wherein“preventing” or “prevention” is to be understood to refer to themanagement and care of a patient for the purpose of hindering thedevelopment of the condition, disease or disorder, and includes theadministration of the pharmaceutical compositions to prevent the onsetof symptoms or complications. The individual to be treated is a humanbeing. An individual to be treated according to the present inventioncan be of various ages and can be both female and male.

The term “thrombomodulin” or TM or CD141 or BDCA-3, is an integralmembrane protein expressed on the surface of endothelial cells servingas a cofactor for thrombin. It reduces blood coagulation by convertingthrombin to an anticoagulant enzyme from a procoagulant enzyme. It isencoded by the THBD gene.

The term “soluble thrombomodulin” or “sTM” refers to a soluble form ofthrombomodulin present in blood and other body fluids in human. A highcirculating level of soluble thrombomodulin in the blood may eitherreflect increased shedding or release of thrombomodulin from theendothelium or reduced metabolism or excretion (the latter is typicalfinding in patients with chronic kidney disease). A low circulatinglevel of thrombomodulin reflects that the shedding or release ofthrombomodulin is low/normal.

The term “thrombomodulin level” used herein refers to the level ofthrombomodulin in the circulating blood of a human, including the levelin whole blood, plasma or serum. A given thrombomodulin level measuredin plasma with the result ng/ml plasma, may not correspond to thethrombomodulin level measured in whole blood with the result ng/ml wholeblood as the plasma fraction of whole blood is only approximately 55%.

The term “high thrombomodulin” (FIG. 7) refers to a plasma concentrationof thrombomodulin above 4 ng/ml in a blood sample drawn from an acutecritically ill patient earliest possible. With earliest possible meansthat the blood sample should be drawn minutes to hours after theinjurious hit either pre-hospital, at hospital admission or uponoccurrence of the acute critical illness in-hospital. Thrombomodulinreveals an area under the receiver operating characteristic (ROC) curveof 0.744 (0.712-0.776) in predicting 28-day mortality. The level 4 ng/mlreveals a sensitivity (true-positive rate) of 0.781 and a 1-specificity(false-positive rate) of 0.407 and the highest possible Youdens Index (asingle statistic parameter that captures the performance of a diagnostictest).

Choosing a lower threshold level of thrombomodulin results in increasedtrue-positive rate (sensitivity) and false-positive rate(1-specificity); whereas choosing a higher threshold level ofthrombomodulin results in reduced true-positive rate (sensitivity) andfalse-positive rate (1-specificity) (Table 3). Both a lower or higherthreshold level of thrombomodulin will thus result in a lower YoudenIndex and hence lower performance of the diagnostic test.

It should be noted that a thrombomodulin level of 4 ng/ml in plasma maynot correspond to the thrombomodulin level measured in whole blood sincethe plasma fraction of whole blood is only approximately 55%.

The term “low thrombomodulin” (FIG. 7) refers to a plasma concentrationof thrombomodulin below 4 ng/ml in a blood sample drawn from an acutecritically ill patient earliest possible. With earliest possible meansthat the blood sample should be drawn minutes to hours after theinjurious hit either pre-hospital, at hospital admission or uponoccurrence of the acute critical illness in-hospital. A lowerthrombomodulin level in an acute critically ill patient is associatedwith an increased change of a good clinical outcome.

The term “standard care” (FIG. 7) refers to but is not limited to thenormal care and/or treatment a patient receives in the hospital such asventilation therapy (e.g. mechanical ventilation) , hemodialysis,vasopressor therapy, fluid therapy, blood transfusion therapy withadministration of red blood cell concentrates, fresh frozen plasma,platelet concentrates, whole blood or coagulation factor concentrates,systemic antibiotic and/or antiviral and/or antifungal and/orantiprotozoic therapy, parenteral nutrition, granulocyte infusion, Tcell infusion, stem cell infusion, anticoagulant and/or antithrombotictherapy including low molecular weight heparins, administration ofcorticosteroids, tight glycemic control etc.

The term “early treatment” (FIG. 7) refers herein to early as possibletreatment with prostacyclin or analogues thereof optimally alreadypre-hospital or within minutes or hours after admission to the hospitalor within minutes or hours upon the occurrence of an in-hospitalinjurious hit. The i.v.

infusion with prostacyclin or analogues thereof in the dose should beginas early as possible after the thrombomodulin test results has becomeavailable i.e., within 30 min-6 hours after known test results,pre-hospital as well as in-hospital.

The term “lower T-level” (FIG. 7) refers to a lower-than-baseline levelof thrombomodulin where baseline refers to the first initial time-pointwhere thrombomodulin is measured. Thus, at all time-points post-baselinewhere thrombomodulin is measured, the decision to continue or ceaseprostacyclin treatment for the following 24 hours depends on the currentthrombomodulin level as compared to the baseline thrombomodulin level.The therapy is thus personalized for the individual patient.

The term “improved CC” (FIG. 7) or “improved clinical condition” refersto an improvement in the clinical condition of the patient and/or areduction in patient requirement for organ supportive care, which iseither based on the attending doctors/medical staffs impression or onmore objective criteria such as disease severity scores like, but notlimited to, Sequential Organ Failure Assessment (SOFA) score. Areduction in requirement for organ supportive care could be but is notlimited to reduced need for vasopressor treatment, reduced need forventilator support and reduced need for oxygen, reduced need fordialysis etc.

The term “continue prostacyclin and standard care” (FIG. 7) means thatthe prostacyclin treatment should continue for another 24 hours inaddition to stand care. This decision is based on the thrombomodulinlevel and the clinical condition of the patient.

The term “pre-hospital” refers to the phase before the patient reachesthe hospital either at the scene of the accident or injurious hit (whilethe patients receives lifesaving emergency care treatment or isstabilized for transportation to the hospital) or during transportationto the hospital (by car, helicopter, plain, boat, train etc.).Pre-hospital treatment refers to any treatment being initiated beforethe patient reaches the hospital.

The term “individual” refers to a human subject.

The term “patient” refers to a human subject that is ill and/or requiresmedical treatment.

The term “personalized medicine” refers herein to an individualizedtreatment where patients with high thrombomodulin levels are treatedwith prostacyclin or analogues thereof whereas patients with lowthrombomodulin levels (whom may look similar clinically to patients withhigh thrombomodulin levels) are not treated with prostacyclin oranalogues thereof.

The term “prostacyclin” refers to the lipid molecule prostacyclin(PGI₂), which is a member of the eicosanoids family. The definition asused herein also includes prostacyclin analogs, prostacyclin variants orprostacyclin receptors agonists which have affinity for prostacyclinreceptors and may be able to mediate functions similar to the functionsmediated by prostacyclin i.e., the prostacyclin analogs or variants arefunctional equivalents of prostacyclin.

The terms “analogue” or “variant” are meant as any analogue or variantof a compound capable of treating a certain condition of the human body,particularly analogs and/or variants of prostacyclin which arefunctional equivalents of the compound.

The term “prostacyclin analogue”, “prostacyclin variant” or“prostacyclin receptor agonist” have affinity for prostacyclin receptorsand may be able to mediate functions similar to the functions mediatedby prostacyclin i.e., the prostacyclin analogs, prostacyclin variantsand prostacyclin receptor agonists are functional equivalents ofprostacyclin. These compounds include but are not limited to Iloprost,Epoprostenol, Epoprostenol Sodium (flolan), Selexipag, Beraprost,Beraprost Sodium, Treprostinil, Treprostenil Sodium, PegylatedTreprostinil, Treprostinil Diethanolamine,2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide (long acting prostacyclin receptor agonist prodrug),{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}acetic acid,8-[1,4,5-triphenyl-1H-imidazol-2-yl-oxy]octanoic acid (IP receptoragonist), carbacyclin (prostacyclin analog), isocarbacyclin(prostacyclin analog), cicaprost (prostacyclin analog),7,8-dihydro-5-(2-(1-phenyl-1-pyrid-3-yl-methiminoxy)-ethyl)-a-naphthyloxyaceticacid (IP receptor-specific non-prostanoid),2-[3-[2-(4,5-diphenyl-2-oxazolyl)ethyl] phenoxy]acetic acid(non-prostanoid prostacyclin partial agonist),[3-[4-(4,5-diphenyl-2-oxazolyl)-5-oxazolyl] phenoxy]acetic acid(non-prostanoid prostacyclin partial agonist), 17[alpha],20-dimethyl-[DELTA]6,6a-6a-carba PGI1 (non-prostanoid prostacyclinpartial agonist),15-deoxy-16[alpha]-hydroxy-16[beta],20-dimethyl-[DELTA]6,6a-6a-carbaPGI1 (non-prostanoid prostacyclin partial agonist).

The terms “prostacyclin analogue”, “prostacyclin variant” or“prostacyclin receptor agonist” refer to a drug that can initiate aphysiologic or a pharmacologic response characteristic of prostacyclin.Prostacyclin analogs, prostacyclin variants and prostacyclin receptoragonists according to the present invention includes, but are notlimited to, compounds that have affinity for the prostacyclin receptorand are capable of activating a prostacyclin receptor response in amanner similar to prostacyclin.

The term “protection” refers to reversal, reduction, ameliorating,alleviating or relieving degradation of the endothelium and itsglyocalyx, the endothelium and/or glycocalyx themselves; delaying theprogression of the endothelial cell damage, glycocalyx degradation;increasing endothelial cell rejuvenation and increasing the productionof the glycocalyx and components of the glycocalyx, and/or reducing therisk of, or preventing the damage to endothelial cells and degradationof the glycocalyx.

The term “endothelial modulating effects” is intended to meanpharmacological treatment aiming at maintaining or bringing theendothelium into a quiescent inactivated, antiadhesive, anticoagulantand anti-inflammatory state hereby preserving, restoring or promotingvascular integrity and normal functional responsiveness of theendothelium.

The term “endothelial modulator” encompasses any agent or compound thatinfluences the endothelium to either maintain or develop into a statewhich optimally preserves and ensures vascular integrity and normalfunctional responsiveness. In a state with maintained vascularintegrity, the endothelium also exerts quiescent inactivated,antiadhesive, anticoagulant and anti-inflammatory properties.

The term “dose” as used herein means a dose sufficient to produce thedesired effect in relation to the conditions for which it isadministered, in particular an amount of a compound capable ofmodulating, protecting and/or treating the endothelium and hereby treatsystemic endotheliopathic syndrome. Normally the dose should be capableof preventing or lessening the severity or spread of the condition orindication being treated. The exact dose will depend on thecircumstances, such as the condition being treated, the administrationschedule, the half-life of the compound capable of modulating,protecting and/or treating the endothelium and the general health of thesubject.

The term “thrombelastography” or “TEG” refers to a commerciallyavailable viscoelastic whole blood hemostasis assay employing citratedor non-anticoagulated blood. The TEG analyzer uses a small whole bloodsample in a rotating cup and a pin suspended in the blood by a torsionwire, which is monitored for motion. To speed up the clot formation, astandardized amount of an activator of coagulation (e.g. kaolin, tissuefactor) may be added to the cup just before the pin is placed in thecup. The TEG assay is suitable for determining important parameters inthe clotting activity (clot initiation time, clot build up dynamics),clot strength (maximum amplitude (MA)) and clot degradation(fibrinolysis). The TEG system's approach to monitoring patienthemostasis is based on the premise that the end result of the hemostaticprocess is the clot. The clot's physical properties determine whetherthe patient will have normal hemostasis, or will be at increased riskfor hemorrhage or thrombosis.

The term “Multiplate” refers to a commercially available impedanceaggregometry based whole blood platelet function analyzer employinganti-coagulated blood. The Multiplate analyzer uses a small whole bloodsample in a chamber with two pairs of platinum electrodes. Standardizedplatelet agonists are added to the blood in the chamber to investigatespecific platelet activation/signaling pathways. The Multiplate assay isbased on the attachment of platelets on two platinum electrodes,resulting in an increase of electrical resistance between theelectrodes. The change of resistance (called impedance) is continuouslyrecorded and is proportional to the amount of platelets sticking to theelectrodes and hence proportional to platelet aggregation.

The term “antiadhesive” refers to the effect of compound(s) that reducesthe platelets ability to adhere to the endothelium and ultimately formthrombi.

The term “anticoagulant” refers to the effect of compound(s) thatreduces or inhibits pro-coagulant coagulation factor activity in theblood and hence reduces or inhibits coagulation of the blood.

The term “antithrombotic” refers to the effect of compound(s) thatreduces the platelets ability to aggregate and adhere and interact inthe clot building process and hence form thrombi.

The term “antiaggregatory” refers to the effect of compound(s) thatreduces the platelets ability to aggregate and interact in the clotbuilding process and hence form thrombi.

The term “coagulopathy” (also called impaired clotting ability, clottingdisorder or bleeding disorder) is any defect in the body's mechanism forcoagulation and clot building, causing a predisposition either for tooslow (hypocoagulability) or too quick (hypercoagulability) clotformation. Clinically, coagulopathy can present with both increasedbleeding tendency and increased thromboembolic events/increased risk ofthrombosis.

The term “glycocalyx” refers here to the carbohydrate-rich layer whichis covering the endothelial cells in healthy individuals. The glycocalyxcomprises proteoglycans which can be soluble or linked to theendothelial cell membrane.

The term “organ failure” refers to an altered organ function in anacutely ill patient requiring medical intervention to achieve bodyhomeostasis and/or to compensate for the loss of function from thatfailing organ. The organs include but are not limited to heart andvessels (cardiac failure, vascular collapse, hypotension, organfailure), lungs (respiratory failure), liver (liver failure), kidneys(renal failure), brain (encephalopathy).

The term “multiple organ failure” (abbreviated MOF) or “multiple organdysfunction syndrome” (abbreviated MODS) is altered function of morethan one organ in an acutely ill patient requiring medical interventionto achieve homeostasis and/or to compensate for the loss of functionfrom the failing organs.

The term “shedding of thrombomodulin” or “release of thrombomodulin” asused herein refers to the process whereby cell-bound thrombomodulin isremoved from the cell and becomes soluble in the blood and/or plasmaphase of the blood. This event reflects endothelial cell damage.

The term “shedding” of the glycocalyx is herein referred to asdegradation of the glycocalyx and release of its components that herebybecomes soluble in the blood and/or plasma phase of the blood.

The term “reperfusion injury” as used herein refers to damage to tissuecaused when blood supply returns to the tissue after a period ofischemia. The absence of oxygen and nutrients from blood creates acondition where the restoration of circulation results in inflammationand oxidative damage through the induction of oxidative stress ratherthan restoration of normal function.

The term “fibrinolysis” means a process wherein a fibrin clot, theproduct of coagulation, is broken down.

The term “sepsis” is used in the conventional clinical meaning,referring to a whole-body inflammatory state (called systemicinflammatory response syndrome (SIRS)) AND the presence of a known orsuspected infection. “Severe sepsis” is defined as sepsis-induced organdysfunction or tissue hypoperfusion (manifesting e.g. as hypotension,elevated lactate, decreased urine output or altered mental status).“Septic shock” is severe sepsis plus persistently low blood pressuredespite the administration of intravenous fluids. Sepsis can lead tosevere sepsis, septic shock, multiple organ dysfunctionsyndrome/multiple organ failure (MODS/MOF) and death.

The term “systemic inflammatory response syndrome” or “SIRS” is used inthe conventional clinical meaning, referring to systemic inflammation inresponse to an insult without confirmed infectious process. SIRS can bediagnosed when 2 or more of the following criteria are present: 1) Bodytemperature less than 36° C. (96.8° F.) or greater than 38° C.(100.4°F.); 2) Heart rate greater than 90 beats per minute; 3) Tachypnea (highrespiratory rate), with greater than 20 breaths per minute or anarterial partial pressure of carbon dioxide less than 4.3 kPa (32 mmHg)and 4) White blood cell count less than 4000 cells/mm³ (4×10⁹ cells/L)or greater than 12,000 cells/mm³ (12×10⁹ cells/L) or the presence ofgreater than 10% immature neutrophils (band forms). When an infection issuspected or proven (by culture, stain, or polymerase chain reaction(PCR)), together with SIRS, this is per definition sepsis.

The term “systemic inflammation” is altered organ function in an acutelyill patient due to the nonspecific conserved response of the body(vasculature, immune system, tissues) to infections, non-infectiousantigens, trauma, burn, organ/tissue destruction/degeneration/damage,ischemia, haemorrhage, intoxication, and/or malignancy.

The term “ELISA” refers to “Enzyme linked immunosorbent assay”. In theELISA method, bound antigen/antibody (Ab) is detected by an antibodylinked (primarily or secondarily) to an enzyme (Ab*) whose activity canbe determined. The activity of the Ab* serves as a quantitative estimateof the amount of the investigated antigen/antibody in the biologicalspecimen.

The term “Scoring systems” refers to scoring systems developed tostandardize the evaluation of a patient's prognosis (risk of death),disease severity or disease progression. Depending on the score, it isapplied at admission, during ICU/hospital stay and/or at specifictime-points after admission (e.g. after 6 months). The scores thusprovide standardized assessments of mortality risk, morbidity, diseaseseverity, clinical disease progression and outcome.

The term “Injury Severity Score” or “ISS” refers to an anatomicalscoring system that provides an overall score for trauma patients withmultiple injuries. Each injury is assigned an Abbreviated Injury Scale(AIS) score and is allocated to one of six body regions (Head, Face,Chest, Abdomen, Extremities (including Pelvis), External). Only thehighest AIS score in each body region is used. To calculate the ISSscore, the 3 most severely injured body regions have their score squaredand added together to produce the ISS score: AlSx²+AlSy²+AlSz²=ISS. TheISS score takes values from 0 to 75. If an injury is assigned an AIS of6 (unsurvivable injury), the ISS is automatically assigned to 75. TheISS is virtually the only anatomical scoring system in use andcorrelates linearly with mortality, morbidity, hospital stay and othermeasures of severity. The ISS is applied one time at admission but notat later time-points during hospital stay.

The term “Glasgow Coma Scale” or “GCS” score refers to a neurologicalscoring scale that in an objective way records the conscious state of apatient for initial as well as subsequent assessment. It was initiallyused to assess the level of consciousness after head injury but thescale is now used by first aid, emergency medical services (EMS), nursesand doctors as being applicable to all acute medical and traumapatients. In hospitals it is also used in monitoring patients while inthe intensive care unit (ICU). The GCS is applied at admission andhereafter at any time-point during hospital stay for evaluation of thepresent level of or changes in consciousness.

The GCS is scored between 3 and 15 (the sum of points given for each ofthe three evaluation parameters), 3 being the worst, and 15 the best. AGCS of 13 or higher correlates with a mild brain injury, 9-12 is amoderate injury, ≤8 is a severe brain injury 3 is deep unconsciousness.

GCS is composed of three parameters: Best Eye Response, Best VerbalResponse, and Best Motor Response:

Best Eye Response 1. No eye opening (max 4 points) 2. Eye opening topain 3. Eye opening to verbal command 4. Eyes open spontaneously BestVerbal Response 1. No verbal response (max 5 points) 2. Incomprehensiblesounds 3. Inappropriate words 4. Confused 5. Orientated Best MotorResponse 1. No motor response (max 6 points) 2. Extension to pain 3.Flexion to pain 4. Withdrawal from pain 5. Localizing pain

The term “Simplified Acute Physiology Score II” or “SAPS II” refers to ascoring system designed to measure the severity of disease for patients5 years admitted to Intensive Care Units (ICU).

The measurement has to be completed 24 hours after admission to the ICU,resulting in an integer point score (sum of worst value sub-scores)between 0 and 163 and a predicted mortality between 0% and 100%(mortality corresponding to 10%, 25%, 50%, 75% and 90% in patients witha score or 29, 40, 52, 64 and 77, respectively). No new score can becalculated during the ICU stay, but if a patient is discharged from theICU and readmitted, a new SAPS II score can be calculated.

The score is based on the worst value of the following physiologicparameters within the past 24 hours:

Parameter Unit Score Age years Heart Rate bpm Systolic Blood PressuremmHg Temperature ° C. or F. Glasgow Coma Scale score MechanicalVentilation yes, no or CPAP PaO₂ mmHg FiO₂ % Urine Output ml Blood UreaNitrogen mg/dl Sodium mEq/l Potassium mEq/l Bicarbonate mEq/l Bilirubinmg/dl White Blood Cell ×10⁹/l Chronic diseases metastatic cancer,hematologic malignancy or AIDS Type of admission scheduled surgical,medical or unscheduled surgical Sum X points

The term “Sequential Organ Failure Assessment” or “SOFA” score refers toa scoring system applied to determine the extent of a person's organfunction or degree of organ failure. The score is based on six differentsub-scores, one each for the respiratory, cardiovascular, hepatic,coagulation, renal and neurological systems. The point given for eachsub-score is summed up to give the total SOFA score. The score istypically determined at admission to ICU and hereafter daily to evaluatedisease progression. The score is applicable to all types of ICUpatients including trauma, sepsis, resuscitated cardiac arrest patients,major surgery etc.

Organ SOFA sup-score points system Parameter 1 2 3 4 Nervous GCS 13-1410-12 6-9  <6 Respiratory PaO2/FiO2 <400 <300 <200 AND <100 AND [mmHg]ventila-tor ventilator Cardiovascular MAP [mmHg] MAP < 70 dop ≤ 5 ORdop > 5 OR dop > 15 OR and/or dob (any epi ≤ 0.1 OR epi > 0.1 ORvasopressor dose) nor ≤ 0.1 nor > 0.1 (dop, epi, nor) Renal Creatinine110-170 171-299 300-440 >440 [μmol/l] or OR <500 ml/d OR <200 ml/d urineoutput Liver Bilirubin >20-32  >33-101 >102-204  >204 [μmol/l]Coagulaion Platelet <150 <100 <50  <20 count [10⁹/l]

The term “Cerebral Performance Category” or “CPC” refers to a scale todetermine neurological outcome after cardiac arrest. The CPC is appliedat any time after the cardiac arrest at a minimum of standardizedtime-points. The patient is scored from 1-5 depending on the clinicalpresentation.

CPC 1 Good cerebral performance: conscious, alert, able to work, mighthave mild neurologic or psychological deficit CPC 2 Moderate cerebraldisability: conscious, sufficient cerebral function for independentactivities of daily life. Able to work in sheltered environment. CPC 3Severe cerebral disability: conscious, dependent on others for dailysupport because of impaired brain function. Ranges from ambulatory stateto severe dementia or paralysis. CPC 4 Coma CPC 5 Brain death: apnea,areflexia, EEG silence, etc.

The term “Modified Rankin Scale” or “mRS” refers to a commonly usedscale for measuring the degree of disability or dependence in the dailyactivities of people who have suffered a stroke, cardiac arrest, orother causes of neurological disability. The patient is scored from 1-6depending on the clinical presentation.

mRS 1 No symptoms at all mRS 2 No significant disability. Able to carryout all usual activities, despite some symptoms. mRS 3 Slightdisability. Able to look after own affairs without assistance, butunable to carry out all previous activities. mRS 4 Moderate disability.Requires some help, but able to walk unassisted. mRS 5 Severedisability. Requires constant nursing care and attention, bedridden,incontinent. mRS 6 Dead

The term “APACHE” or “APACHE II score”, refers to Acute Physiology andChronic Health Evaluation (APACHE) II score. APACHE II is a scoringsystem designed to measure the severity of disease for patients ≥15years admitted to Intensive Care Units (ICU). The measurement must becompleted 24 hours after admission to the ICU, resulting in an integerpoint score (based on the worst value sub-scores) between 0 and 71.Higher scores correspond to more severe disease and a higher risk ofdeath. The APACHE II score cannot be directly converted to a percentrisk of mortality but can be used to calculate a mortality risk if thepatient's indication for ICU admission is accounted for. No new scorecan be calculated during the ICU stay but if a patient is dischargedfrom the ICU and readmitted, a new APACHE II score can be calculated.The score is based on the worst value of the following physiologicparameters within the past 24 hours:

Parameter Unit Score Age Years Glasgow Coma Scale Score Temperature ° C.of F. Mean Arterial Blood mmHg (MAP) Pressure Heart Rate bpm RespiratoryRate bpm FiO₂ % PaO₂ mmHg Sodium mEq/l Potassium mEq/l Creatinine Mg/dlAcute Renal Failure yes, no Hematocrit % White Blood Cell ×10⁹/l Severeorgan system Liver: Biopsy-proven cirrhosis with insufficiency or isportal hypertension; episodes of past immunocompromised? upper GIbleeding attributed to portal hypertension; or prior episodes of hepaticfailure, encephalopathy, or coma. Cardiovascular: New York HeartAssociation (NYHA) class IV heart failure. Respiratory: Chronicrestrictive, obstructive, or vascular disease resulting in severeexercise restriction (ie, unable to climb stairs or perform householdduties); documented chronic hypoxia, hypercapnia, secondarypolycythemia, severe pulmonary hypertension (>40 mmHg); or respiratordependency. Renal: Receiving chronic dialysis. Immunocompromised: Thepatient has received therapy that suppresses resistance to infection(e.g., immune- suppression, chemotherapy, radiation, long-term orhigh-dose steroids, or advanced leukemia, lymphoma, or AIDS) SUM Xpoints

SUMMARY

The embodiments are disclosed in the present description, drawings andin the claims. In particular, embodiments disclosed in the examples ofthe present disclosure are considered particularly preferred.

The aspects of the disclosed embodiments relate to a method ofdiagnosing, uses of thrombomodulin as a biological marker, a method oftreatment; prostacyclin or an analogue thereof for use in noveltreatments and compositions comprising prostacyclin or an analoguethereof for use in novel treatments, all according to the embodimentsdescribed in this section in particular, and throughout the presentdisclosure.

In a first aspect and implementation there is disclosed a method ofdiagnosing for an individual diagnosed with an acute critical illness,if the individual is a candidate for combination treatment of standardcare for the acute critical illness in combination with administrationof prostacyclin or an analogue thereof, the method comprising:

-   -   measuring a baseline concentration of soluble thrombomodulin in        blood or plasma of the individual;    -   determining if the baseline concentration of soluble        thrombomodulin in blood or plasma is above a threshold level of        at least 2.5 ng/ml; and    -   diagnosing the individual as a candidate for combination        treatment if the baseline concentration is above the threshold        level.

In a second implementation of the first aspect and implementation thereis disclosed a method according to the first aspect and implementationwherein the threshold level is at least 4 ng/ml.

In a third implementation of the first aspect and implementation thereis disclosed a method according to either the first or the secondimplementations of the first aspect wherein the method is a method ofdiagnosing severe endothelial damage in the individual diagnosed withthe acute critical illness; wherein the individual is diagnosed withsevere endothelial damage in addition to the acute critical illness, ifthe baseline concentration is above the threshold level.

In a fourth implementation of the first aspect and implementation thereis disclosed a method according to any of the first to thirdimplementations wherein the method is a method of diagnosing systemicendotheliopathic syndrome in an individual diagnosed with an acutecritical illness; wherein the individual is diagnosed with systemicendotheliopathic syndrome when the individual is diagnosed with both anacute critical illness and severe endothelial damage.

In a second aspect and first implementation thereof there is disclosedthe use of thrombomodulin as a biological marker in a method ofdiagnosing for an individual diagnosed with an acute critical illness,if the individual is a candidate for combination treatment with standardcare for the acute critical illness in combination with administrationof prostacyclin or an analogue thereof, the method comprising:

-   -   measuring a baseline concentration of soluble thrombomodulin in        blood or plasma of the individual;    -   determining if the baseline concentration of soluble        thrombomodulin in blood or plasma is above a threshold level of        at least 2.5 ng/ml; and    -   diagnosing the individual as a candidate for combination        treatment if the baseline concentration is above the threshold        level.

In a second implementation of the second aspect and first implementationthereof there is disclosed a use according to the second aspect andfirst implementation thereof wherein the threshold level is at least 4ng/ml.

In a third implementation of the second aspect and first implementationthereof there is disclosed a use according to either the first or secondimplementations of the second aspect wherein the method is a method ofdiagnosing severe endothelial damage in the individual diagnosed withthe acute critical illness; wherein the individual is diagnosed withsevere endothelial damage in addition to the acute critical illness, ifthe baseline concentration is above the threshold level.

In a fourth implementation of the second aspect and first implementationthereof there is disclosed a use according to any of the first to thirdimplementations of the second aspect wherein the method is a method ofdiagnosing systemic endotheliopathic syndrome in an individual diagnosedwith an acute critical illness; wherein the individual is diagnosed withsystemic endotheliopathic syndrome when the individual is diagnosed withboth an acute critical illness and severe endothelial damage.

In a third aspect and first implementation thereof there is disclosed amethod of treating an acute critical illness in an individual diagnosedwith the acute critical illness and concurrent increase in a measuredthrombomodulin level in blood or plasma of the individual, the methodcomprising:

-   -   (a) measuring a baseline concentration of soluble thrombomodulin        in blood or plasma of the individual;    -   (b) determining if the baseline concentration of soluble        thrombomodulin in blood or plasma is above a threshold level of        at least 2.5 ng/ml;    -   (c) administering a dose of 0.5-4 ng/kg/min of prostacyclin or        an analogue thereof to the individual continuously for a first        time period if the baseline concentration of soluble        thrombomodulin in blood or plasma is above the threshold level        of at least 2.5 ng/ml;    -   (d) measuring at the end of the first time period a        concentration of soluble thrombomodulin in blood or plasma of        the individual;    -   (e) determining if the concentration of soluble thrombomodulin        is lower by at least a decrease of 10% compared to the baseline        concentration of thrombomodulin determined prior to initiation        of the prostacyclin administration;    -   (f) assessing if a clinical improvement of the acute critical        illness in the individual has occurred during the first time        period; and    -   (g) if both a concentration reduction and a clinical improvement        is observed, ceasing prostacyclin administration while        continuing standard care for the acute critical illness; or    -   (h) otherwise continue prostacyclin administration for a second        time period not exceeding the first time period    -   wherein the steps (d) to (h) are repeated until ceasing        prostacyclin administration to the individual following step        (g).

In a second implementation of the third aspect and first implementationthereof there is disclosed a method according to the firstimplementation wherein the threshold level is at least 4 ng/ml.

In a third implementation of the third aspect and first implementationthereof there is disclosed a method according to either the first orsecond implementations wherein the dose is 1-2 ng/kg/min.

In a fourth implementation of the third aspect and first implementationthereof there is disclosed a method according to any of the first tothird implementations wherein the analogue of prostacyclin is eitherIloprost or Flolan.

In a fifth implementation of the third aspect and first implementationthereof there is disclosed a method according to any of the first tofourth implementations wherein the first time period is at least 48hours or at least 72 hours.

In a sixth implementation of the third aspect and first implementationthereof there is disclosed a method according to any of the first tofifth implementations wherein the second time period is 12 hours or 24hours.

In a seventh implementation of the third aspect and first implementationthereof there is disclosed a method according to any of the first tosixth implementations wherein in step (e) the decrease is at least 20%.

In an eight implementation of the third aspect and first implementationthereof there is disclosed a method according to any of the first toseventh implementations wherein the baseline concentration is measuredimmediately after or shortly after recognition of the acute criticalillness.

In a ninth implementation of the third aspect and first implementationthereof there is disclosed a method according to the first to eightimplementations wherein the baseline concentration is measured within 5minutes and up to 6 hours after recognition of the acute criticalillness.

In a tenth implementation of the third aspect and first implementationthereof there is disclosed a method according to any of the first toninth implementations wherein the prostacyclin administration isinitiated immediately or shortly after a completion of step (b) hasdetermined that the baseline concentration of soluble thrombomodulin inblood or plasma is above the threshold level.

In an eleventh implementation of the third aspect and firstimplementation thereof there is disclosed a method according to thetenth implementation wherein the prostacyclin administration isinitiated within 5 min to 12 hours after a completion of step (b) hasdetermined that the baseline concentration of soluble thrombomodulin inblood or plasma is above the threshold level.

In a twelfth implementation of the third aspect and first implementationthereof there is disclosed a method according to any of the first toeleventh implementations wherein prostacyclin or an analog thereof isadministered as according to step (c) prior to steps (a) and (b) havingbeen completed.

In a thirteenth implementation of the third aspect and firstimplementation thereof there is disclosed a method according to any ofthe first to twelfth implementations wherein the concurrent increase ina measured thrombomodulin level in blood or plasma of the individual isthe result of severe endothelial damage.

In a fourteenth implementation of the third aspect and firstimplementation thereof there is disclosed a method according to any ofthe first to thirteenth implementations wherein the concurrent increasein a measured thrombomodulin level in blood or plasma of the individualis the result of systemic endotheliopathic syndrome.

In a fifteenth implementation of the third aspect and firstimplementation thereof there is disclosed a method according to any ofthe first to fourteenth implementations wherein the acute criticalillness is selected from trauma, burn injury trauma, sepsis, severesepsis, septic shock, acute myocardial infarction, cardiac arrest,systemic inflammatory response syndrome (SIRS), acute major surgery,anti-shock therapy, or a thromboembolic event.

In a sixteenth implementation of the third aspect and firstimplementation thereof there is disclosed a method according to any ofthe first to fifteenth implementations wherein prostacyclinadministration is by intravenous infusion.

In a fourth aspect and first implementation thereof there is disclosedprostacyclin or an analogue thereof for use in the treatment of an acutecritical illness, the treatment according to any of the first tosixteenth implementations of the third aspect.

In a fifth aspect and first implementation thereof there is disclosed acomposition comprising prostacyclin or an analogue thereof for use inthe treatment of an acute critical illness, the treatment according toany of the first to sixteenth embodiments of the third aspect.

In a sixth aspect and a first implementation thereof there is discloseda method of diagnosing for an individual diagnosed with an acutecritical illness, if the individual is a candidate for combinationtreatment of standard care for the acute critical illness in combinationwith administration of prostacyclin or an analogue thereof, the methodcomprising:

-   measuring a baseline concentration of syndecan-1 in blood or plasma    of the individual;-   determining if the baseline concentration of syndecan-1 in blood or    plasma is above a threshold level of at least 40 ng/ml; and-   diagnosing the individual as a candidate for combination treatment    if the baseline concentration is above the threshold level.

In a second implantation of the sixth aspect the threshold level forsyndecan-1 is at least 60 ng/ml.

In a seventh aspect and a first implementation thereof there isdisclosed a method of treating an acute critical illness in anindividual diagnosed with the acute critical illness and concurrentincrease and a measured syndecan-1 level in blood or plasma of theindividual, the method comprising:

-   (a) measuring a baseline concentration of syndecan-1 in blood or    plasma of the individual;-   (b) determining if the baseline concentration of syndecan-1 in blood    or plasma is above a threshold level of at least 40 ng/ml;-   (c) administering a dose of 0.5-4 ng/kg/min of prostacyclin or an    analogue thereof to the individual continuously for a first time    period if the baseline concentration of syndecan-1 in blood or    plasma is above the threshold level of at least 40 ng/ml;-   (d) measuring at the end of the first time period a concentration of    soluble thrombomodulin in blood or plasma of the individual;-   (e) determining if the concentration of soluble thrombomodulin is    lower by at least a decrease of 10% compared to a measured baseline    concentration of thrombomodulin determined prior to initiation of    the prostacyclin administration;-   (f) assessing if a clinical improvement of the acute critical    illness in the individual has occurred during the first time period;    and-   (g) if both a concentration reduction and a clinical improvement is    observed, ceasing prostacyclin administration while continuing    standard care for the acute critical illness; or-   (h) otherwise continue prostacyclin administration for a second time    period not exceeding the first time period-   wherein the steps (d) to (h) are repeated until ceasing prostacyclin    administration to the individual following step (g).

In a second implantation of the seventh aspect the threshold level forsyndecan-1 is at least 60 ng/ml.

In an eighth aspect and a first implementation thereof there isdisclosed a method of diagnosing for an individual diagnosed with anacute critical illness, if the individual is a candidate for combinationtreatment of standard care for the acute critical illness in combinationwith administration of prostacyclin or an analogue thereof, the methodcomprising:

-   measuring a baseline concentration of adrenaline in blood or plasma    of the individual;-   determining if the baseline concentration of syndecan-1 in blood or    plasma is above a threshold level of at least 225 pg/ml; and-   diagnosing the individual as a candidate for combination treatment    if the baseline concentration is above the threshold level.

In a second implantation of the eighth aspect the threshold level foradrenaline is at least 300 pg/ml.

In a ninth aspect and a first implementation thereof there is discloseda method of treating an acute critical illness in an individualdiagnosed with the acute critical illness and concurrent increase and ameasured adrenaline level in blood or plasma of the individual, themethod comprising:

-   (i) measuring a baseline concentration of adrenaline in blood or    plasma of the individual;-   (j) determining if the baseline concentration of adrenaline in blood    or plasma is above a threshold level of at least 225 pg/ml;-   (k) administering a dose of 0.5-4 ng/kg/min of prostacyclin or an    analogue thereof to the individual continuously for a first time    period if the baseline concentration of adrenaline in blood or    plasma is above the threshold level of at least 225 pg/ml;-   (l) measuring at the end of the first time period a concentration of    soluble thrombomodulin in blood or plasma of the individual;-   (m) determining if the concentration of soluble thrombomodulin is    lower by at least a decrease of 10% compared to a measured baseline    concentration of thrombomodulin determined prior to initiation of    the prostacyclin administration;-   (n) assessing if a clinical improvement of the acute critical    illness in the individual has occurred during the first time period;    and-   (o) if both a concentration reduction and a clinical improvement is    observed, ceasing prostacyclin administration while continuing    standard care for the acute critical illness; or-   (p) otherwise continue prostacyclin administration for a second time    period not exceeding the first time period-   wherein the steps (d) to (h) are repeated until ceasing prostacyclin    administration to the individual following step (g).

In a second implantation of the ninth aspect wherein the threshold levelfor adrenaline is at least 300 pg/ml.

In a tenth aspect and a first implementation thereof there is discloseda method of treating an acute critical illness in an individualdiagnosed with the acute critical illness and concurrent increase in ameasured thrombomodulin level in blood or plasma of the individual, themethod comprising:

-   (a) measuring a baseline concentration of soluble thrombomodulin in    blood or plasma of the individual;-   (b) determining if the baseline concentration of soluble    thrombomodulin in blood or plasma is above a threshold level of at    least 2.5 ng/ml;-   (c) administering a dose of 0.5-4 ng/kg/min of prostacyclin or an    analogue thereof to the individual;

In an eleventh aspect and a first implementation thereof there isdisclosed a method of treating an acute critical illness in anindividual diagnosed with the acute critical illness and concurrentincrease in a measured syndecan-1 level in blood or plasma of theindividual, the method comprising:

-   (d) measuring a baseline concentration of syndecan-1 in blood or    plasma of the individual;-   (e) determining if the baseline concentration of syndecan-1 in blood    or plasma is above a threshold level of at least 40 ng/ml;-   (f) administering a dose of 0.5-4 ng/kg/min of prostacyclin or an    analogue thereof to the individual;

In a twelfth aspect and a first implementation thereof there isdisclosed a method of treating an acute critical illness in anindividual diagnosed with the acute critical illness and concurrentincrease in a measured adrenaline level in blood or plasma of theindividual, the method comprising:

-   (g) measuring a baseline concentration of adrenaline in blood or    plasma of the individual;-   (h) determining if the baseline concentration of adrenaline in blood    or plasma is above a threshold level of at least 225 pg/ml;-   (i) administering a dose of 0.5-4 ng/kg/min of prostacyclin or an    analogue thereof to the individual;

In a further implementation of any one of the first, second, third andsixth to twelfth aspect the measurement of thrombomodulin, syndecan-1,adrenaline or VEGF is conducted by a point-of-care (POC) assay.

DESCRIPTION OF DRAWINGS AND TABLES

FIG. 1 shows a schematic presentation of the systemic endotheliopathicsyndrome at the vascular level.

FIG. 1 is a schematic presentation of systemic endotheliopathic syndromeillustrating, at the vascular level, how an acute critically ill patientwith an increasing injurious hit will experience progressive damage tothe endothelium resulting in endothelial shedding/release ofthrombomodulin and ensuing increases in circulating solublethrombomodulin (sTM, bubbles). Progressive endothelial damage willclinically result in impaired vascular barrier function, capillaryleakage, bleeding, hypotension, multiple organ failure and ultimatelydeath.

FIGS. 2a-2d are graphs showing test results that illustrate the effectof Flolan® infusion in healthy volunteers.

FIG. 2a shows a graph of a soluble thrombomodulin (ng/ml) at baseline (0h), at the end of (2 h) and after (4 h, 5 h, 6 h, 8 h, 24 h) 2 hours 4ng/kg/min Flolan® infusion in healthy volunteers. *, significantdifference from baseline.

FIG. 2b shows a graph of protein C (%) at baseline (0 h), at the end of(2 h) and after (4 h, 5 h, 6 h, 8 h, 24 h) 2 hours 4 ng/kg/min Flolan®infusion in healthy volunteers. *, significant difference from baseline.

FIG. 2c shows a graph of plasminogen activator inhibitor (PAI)-1 (ng/ml)at baseline (0 h), at the end of (2 h) and after (4 h, 5 h, 6 h, 8 h, 24h) 2 hours 4 ng/kg/min Flolan® infusion in healthy volunteers. *,significant difference from baseline.

FIG. 2d shows a graph of antithrombin (microg/ml) at baseline (0 h), atthe end of (2 h) and after (4 h, 5 h, 6 h, 8 h, 24 h) 2 hours 4ng/kg/min Flolan® infusion in healthy volunteers. *, significantdifferences from baseline.

FIGS. 3a to 3c are graphs showing test results that illustrate theeffect of Ilomedin® infusion in healthy volunteers.

FIG. 3a shows a graph of soluble thrombomodulin (ng/ml) at baseline(baseline), during (30 min), at the end of (2 h) and after (2 h post) 2hours 1 ng/kg/min Ilomedin® infusion in healthy volunteers. *,significant difference from baseline; (*), borderline significantdifference from baseline.

FIG. 3b shows a graph of protein C (%) at baseline (baseline), during(30 min), at the end of (2 h) and after (2 h post) 2 hours 1 ng/kg/minIlomedin® infusion in healthy volunteers. *, significant difference frombaseline; (*), borderline significant difference from baseline.

FIG. 3c shows a graph of prostacyclin (pg/ml) at baseline (baseline),during (30 min), at the end of (2 h) and after (2 h post) 2 hours 1ng/kg/min Ilomedin® infusion in healthy volunteers. *, significantdifference from baseline; ‡, significant difference from previoustime-point.

FIG. 4 is a graph showing test results that illustrates the effect ofIlomedin infusion in patients with acute myocardial infarctionundergoing percutaneous coronary intervention

FIG. 4 shows a graph illustrating soluble E-selectin (ng/ml) at baseline(baseline), at the end of 24 hours (24 h) and after (48 h) 24 hoursactive (0.5 ng/kg/min Ilomedin® infusion) or placebo (0.9% salineinfusion) therapy of AMI PCI patients. ¶, significant difference betweenactive and placebo group.

FIGS. 5a-5c are graphs showing test results that illustrate the effectof Ilomedin infusion in patients undergoing Whipple surgery.

FIG. 5a shows a graph illustrating soluble thrombomodulin (ng/ml)pre-operative (0 h), post-operative (post-op) and 6 hourspost-operatively (6 h) intra- and post-operative active (1 ng/kg/minIlomedin® infusion) or placebo (0.9% saline infusion) therapy of Whipplepatients. *, significant difference from baseline; ‡, significantdifference between active and placebo group.

FIG. 5b shows a graph illustrating nucleosomes (%) pre-operative (0 h),post-operative (post-op) and 6 hours post-operatively (6 h) intra- andpost-operative active (1 ng/kg/min Ilomedin® infusion) or placebo (0.9%saline infusion) therapy of Whipple patients. *, significant differencefrom baseline; ‡, significant difference between active and placebogroup.

FIG. 5c shows a graph illustrating syndecan-1 (ng/ml) pre-operative (0h), post-operative (post-op) and 6 hours post-operatively (6 h) intra-and post-operative active (1 ng/kg/min Ilomedin® infusion) or placebo(0.9% saline infusion) therapy of Whipple patients. *, significantdifference from baseline.

FIG. 6 shows a graph illustrating a receiver operating characteristic(ROC)-curve of thrombomodulin for predicting mortality in acutecritically ill patients suffering from trauma, myocardial infarction,cardiac arrest, sepsis/severe sepsis/septic shock. Thrombomodulin AUCfor predicting 28-day mortality: AUC 0.744 (0.712-0.776), p<0.0001.Highest Youden Index reveals a threshold level of 4.0 ng/mlthrombomodulin in plasma. Sensitivity 0.781, 1-specificity 0.407.

FIG. 7 shows a flow diagram illustrating an example embodiment of amethod for deciding on treatment with prostacyclin or an analoguethereof in patients presenting with acute critically illness incombination with high levels of thrombomodulin.

FIG. 8 shows a flow diagram illustrating the invention as exemplarydescribed in FIG. 7 in terms of its constituent units of identification,treatment and control.

FIG. 9 shows ELISA scores for thrombomodulin in blood versus plasma.Correlations between thrombomodulin concentrations (ng/ml) measured in1:2 diluted plasma (golden standard) and whole blood, either undiluted(FIG. 9a ), diluted 1:2 (FIG. 9b ), or diluted 1:4 (FIG. 9c ).

FIG. 10 shows a graph illustrating a receiver operating characteristic(ROC)-curve of syndecan-1.

FIG. 11 shows a graph illustrating a receiver operating characteristic(ROC)-curve of adrenaline.

FIG. 12 shows Table 1, which details a study of demography and diseaseseverity in ICU patients.

FIG. 13 shows Table 2, which details a study of median thrombomodulinlevels in plasma/serum (ng/ml) in 2,118 acute critically ill patients

FIG. 14 shows Table 3, which details a study of the thrombomodulin levelversus test sensitivity for the patient group of Table 2.

FIG. 15 shows Table 4a, which details results for trauma patients. Table4a details results for injury severity at admission in 635 traumapatients dependent on the level of sTM (soluble thrombomodulin).

FIG. 16 shows Table 4b, which details results based on 270 traumapatients.

FIG. 17 shows Table 5a, which details results for cardiac arrestpatients based on n=169 patients resuscitated from cardiac arrest. FIG.17 Table 5a details the proportion of OHCA patients with sTM>2.5-5-5ng/ml at admission.

FIG. 18 shows Table 5b, which details the absolute and proportional(median) changes in sTM from admission and 24 h and 72 h onward in 28day survivors and non-survivors.

FIG. 19 shows Table 5c, which details the predictive value of sTM atdifferent time-points for poor cognitive outcome (CPC≥3/mRS≥4) evaluatedby ROC analysis.

FIG. 20 shows Table 6, which details results based on 571 STEMI patientson the proportion of OHCA patients with sTM>2.5-5-5 ng/ml at ICUadmission.

FIG. 21 shows Table 7, which details results based on two differentcohorts (n=749 and n=184) of patients with sepsis/severe sepsis/septicshock showing the proportion of septic patients with sTM>2.5-5-5 ng/mlat ICU admission.

FIG. 22 shows Table 8, which details a study of demography data andthrombomodulin levels in plasma and whole blood from 10 healthyvolunteers.

FIGS. 23 and 24 show Table 9 and 10, respectively, which detail thesensitivities and 1-specificities for predicting mortality are obtained(data from 635 trauma patients) when applying other cut-offs than 40ng/ml for syndecan-1 and 225 pg/ml for adrenaline,

DETAILED DESCRIPTION

The aspects of the disclosed embodiments relate to the treatment ofacute critically ill patients. Patients that are acute critically illsuffer from e.g.

severe trauma including burn injury, severe infection including sepsisand septic shock, acute myocardial infarction, resuscitated cardiacarrest, major surgery and/or other conditions that require organsupportive care/intensive care therapy to survive i.e. ventilatortherapy, continuous fluid infusion or blood transfusion, vasopressortherapy etc.

The inventors have discovered that some acute critically ill patients,independent of the type of injurious hit, display evidence of a commonunifying medical condition that is associated with increased mortality.In prior research papers, e.g. [Johansson and Ostrowski, 2010], thepresent inventors have suggested that this common unifying medicalcondition is a distinct diagnosis which the inventors have now labelledsystemic endothelial damage.

Based on their findings in thousands acute critically ill patients, theinventors discovered that a large fraction of these patients presentedwith systemic endothelial damage that across all patients was thestrongest marker for mortality. The combination of acute criticalillness and systemic endothelial damage has been suggested by thepresent inventors to represent a novel disease entity which theinventors now label systemic endotheliopathic syndrome, characterized byimpaired vascular barrier function and capillary leakage, bleeding,hypotension, multiple organ failure and death in acute critically illpatients [Johansson and Ostrowski 2010]. A patient is considered to havesystemic endotheliopathic syndrome when the patient suffers from acutecritical illness AND is diagnosed with systemic endothelial damage.

FIG. 1 illustrates at the vascular level how an acute critically illpatient with an increasing injurious hit will experience progressivedamage to the endothelium resulting in endothelial shedding/release ofthrombomodulin and ensuing increases in circulating solublethrombomodulin (sTM). Progressive endothelial damage will clinicallyresult in impaired vascular barrier function, capillary leakage,bleeding, hypotension, multiple organ failure and ultimately death. Thehypothesized progression of systemic endothelial syndrome is furtherindicated.

The present disclosure demonstrates that systemic endothelial damage canbe identified and diagnosed in a simple manner by using the solublethrombomodulin level in a blood sample as a biological marker for thepresence or absence of systemic endothelial damage. Using thrombomodulinas a biological marker, acute critically ill patients presenting withblood thrombomodulin levels above a given threshold value can be rapidlyidentified as individuals suffering from systemic endothelial damage;thereby in a simple manner differentiating this group of patients fromacute critically ill patients that do not suffer from systemicendothelial damage.

Furthermore, the present disclosure demonstrates that acute criticallyill patients diagnosed with systemic endothelial damage can beneficiallyreceive early treatment with compositions comprising prostacyclin oranalogues thereof, such as prostacyclin (PGI2) and prostacyclin (PGX) oranalogues thereof such as e.g. iloprost, epoprostenol, epoprostenolSodium, treprostenil sodium, selexipag or beraprost; therebysignificantly reducing development of organ failure , improving longterm full recovery and reducing mortality.

A central part of the present invention is a personalized medicineapproach to treatment i.e., that only acute critically ill patientssuffering from systemic endotheliopathic syndrome should be treated withprostacyclin or analogues whereas acute critically ill patients withoutsystemic endotheliopathic syndrome (with low thrombomodulin) should notbe treated with prostacyclin or analogues thereof.

Given the direct effects of prostacyclin and analogs thereof on theendothelium (described in details below), the inventors suggest thatinfusion of low-dose prostacyclin can be used to treat systemicendotheliopathic syndrome.

Proof-of-concept studies of this intervention and its effect on theendothelium measured by changes in circulating levels of thrombomodulinis described below.

The inventors suggest that prostacyclin analogs should be used fortreatment of systemic endotheliopathic syndrome, identified in acutecritically ill patients by a quick test measuring the level ofcirculating thrombomodulin.

In further detail the present invention relates to a method ofdiagnosing, uses of thrombomodulin as a biological marker, a method oftreatment; prostacyclin or an analogue thereof for use in noveltreatments and compositions comprising prostacyclin or an analoguethereof for use in novel treatments, all according to the embodimentsdescribed in this section in particular, and throughout the presentdisclosure.

In a first aspect and embodiment there is disclosed a method ofdiagnosing for an individual diagnosed with an acute critical illness,if the individual is a candidate for combination treatment of standardcare for the acute critical illness in combination with administrationof prostacyclin or an analogue thereof, the method comprising: measuringa baseline concentration of soluble thrombomodulin in blood or plasma ofthe individual; determining if the baseline concentration of solublethrombomodulin in blood or plasma is above a threshold level of at least2.5 ng/ml; and diagnosing the individual as a candidate for combinationtreatment if the baseline concentration is above the threshold level.

This first aspect and embodiment of the invention is further detailed inFIGS. 7 and 8. In FIG. 7, this first aspect and embodiment relates toitems 1 and 2, in FIG. 8 it is illustrated in the column with theheading “Identification”.

Generally, the information contained in FIGS. 7 and 8 is identical andthe skilled reader will realize that in FIG. 7, as mentioned, items 1and 2 correspond to the items of FIG. 8 under the heading“Identification”, whereas the item 3 of FIG. 7 corresponds to the itemsof FIG. 8 under the heading “Treatment”, and the items 4 and 5 of FIG. 7corresponds to the items of FIG. 8 under the heading “Control”.

The advantage of presenting the information of FIGS. 7 and 8 in thesetwo corresponding manners will be immediately obvious to the skilledperson. In FIG. 7 the emphasis is on the sequential nature of the threemain components, “Identification”, “Treatment” and “Control”, which formthe underlying concept of the present invention. However, when the sameinformation is presented as in FIG. 8, it becomes immediately clear tothe skilled person that each of these three concepts, identification,treatment, and control, can be individually manipulated within the scopeof the disclosed embodiments.

As an example, treatment with prostacyclin or an analog thereofaccording to the disclosure of the present invention can be initiatedimmediately upon diagnosis of the patient with an acute criticalillness, without hesitating to obtain the further diagnose of systemicendothelial damage through measurement of the patient's thrombomodulinlevels. This expedites the initiation of the treatment with prostacyclinor an analogue thereof and is possible due to the low risk of an adverseeffect from the mentioned treatment.

When initiating rapid treatment as described above, the firstmeasurement for identification of systemic endothelial damage followingthe disclosure of the present invention, the also becomes the firstcontrol measurement, since a patient which is not in need ofprostacyclin treatment can be stopped from receiving further treatmentonce the first test results have been obtained.

EXAMPLES Studies of the Endothelium in Acute Critically Ill Patients

The inventors have investigated more than 4,400 patients suffering fromsevere trauma, sepsis, acute myocardial infarction, resuscitated cardiacarrest and major surgery, including 1,750 patients with varying degreesof sepsis. In these studies, the inventors found that a common andcritical link may exist between the severity of the injuries;development of coagulopathy and progressive endothelial damage(endotheliopathy) and mortality.

In the above studies, across the different patient groups, the inventorsfound that endothelial cellular damage, evidenced by high circulatinglevels of soluble thrombomodulin, was the strongest marker formortality.

Importantly, across the investigated groups of different patients withacute critical illness, not all patients developed systemicendotheliopathic syndrome and these patients, with low circulatingthrombomodulin, had an excellent outcome with high survival rates. Thiscaused the inventors to realize that in some instances systemicendotheliopathic syndrome may result from acute critical illness such ase.g. severe trauma, sepsis, acute myocardial infarction, resuscitatedcardiac arrest and major surgery or other types of acute criticalillness, but that this outcome is not necessarily given.

Consequently, patients presenting with concurrent acute critical illnessAND high circulating thrombomodulin levels above a given criticalthreshold limit can be diagnosed as presenting with systemicendotheliopathic syndrome.

To the surprise of the inventors they have now further discovered thatpatients presenting with systemic endotheliopathic syndrome, i.e.presenting with an acute critical illness and high (above a giventhreshold) thrombomodulin levels in the blood, will benefit fromtreatment with prostacyclin or analogues thereof with the aim of curingor alleviating the suffered acute critical illness and reducing patientmortality in situations where patients presenting with the same acutecritical illness but not presenting with the additional diagnose ofsystemic endothelial damage as evidence by a high (above a giventhreshold) thrombomodulin level in the blood, will not.

Many acute critically ill patients are in shock i.e. a medical emergencycondition in which the tissue and organs of the body are deprived ofoxygen due to inadequate blood flow.

There are four stages of shock: Initial, compensatory, progressive andrefractory. In the initial stage, hypoperfusion causes hypoxia which isfollowed by compensatory physiological mechanisms, including neural(including sympathoadrenal activation) and hormonal mechanisms in anattempt to reverse the condition. If the condition is not reversed itwill proceed into the progressive stage where the compensatorymechanisms begin to fail resulting in severely reduced cell, tissue andorgan perfusion and a rise in anaerobic metabolism. Eventually this willlead to leakage of fluid and protein into tissues and upon loss of this(intravascular) fluid, the blood concentration and viscosity willincrease, causing sludging of the microcirculation. Also, the prolongedvasoconstriction will cause the vital organs to be compromised due toreduced perfusion. In the final refractory stage, the vital organs willhave failed and the shock can no longer be reversed.

All stages of shock are associated with systemic endothelialperturbations ranging from reversible activation and damage toirreversible extensive injury. Therefore the present inventors haverealized that administration of prostacyclin or an analogue thereof toacute critically ill patients according to the teaching of the presentdisclosure, will serve through its beneficial effects on the vascularendothelium and its cytoprotective effects, as an anti-shock therapy.

Testing and Treatment Acute Critically Ill Patients

Patients that are acute critically ill suffer from e.g. severe trauma,including burn injury, severe infection including sepsis and septicshock, acute myocardial infarction, resuscitated cardiac arrest, majorsurgery and other conditions that happens immediately and require organsupportive care to survive i.e. ventilator therapy, continuous fluidinfusion or blood transfusion, vasopressor therapy etc.

When an acute critically ill patient requires pre-hospital emergencycare OR is admitted to the Hospital (the Emergency Department, theTrauma Center or other Departments that have the initial contact withthe patient) OR when a hospitalized patient becomes acute critically illin-hospital (in hospital cardiac arrest (INCA), in hospital suddendevelopment of septic shock etc.), a blood sample for measurement ofthrombomodulin is drawn as early as possible i.e., during transportationof the patient to the Hospital or within minutes up to hours fromHospital admission or in-hospital recognition of the acute criticalillness. At the same time point the patient receives standard care forthe acute critical illness i.e. ventilation, fluid therapy, bloodtransfusion, antibiotics, vasopressor therapy, damage control surgery,specific drugs used for the specific acute critical illness e.g.platelet inhibitors and anticoagulants in cardiac arrest caused bymyocardial infarction.

Optimally, the patient blood sample is drawn within the first minutes orhours pre-hospital or within the first minutes or hours afteradmission/recognition of acute critical illness. However, blood samplestaken up to 12 hours of admission/recognition of acute critical illnesscan still be relevant.

Thrombomodulin Testing

The whole blood sample is immediately spun in a centrifuge (e.g. 3000rpm for 10 minutes) to separate the plasma phase from the blood cellphase. A small volume of the plasma fraction (e.g. from 10-200 μl) ispipetted onto an immunoassay e.g. an enzyme linked immunosorbent assay(ELISA). Currently available ELISA assays require approximately 1 hincubation with patient sample, hereafter 30 min incubation withdetection reagent and hereafter 15 min incubation with substratesolution before the ELISA plate can be read by an ELISA reader,corresponding to a total assay time of less than 2 hours. Thethrombomodulin concentration in the patient sample is calculated bycomparing the assay output e.g. absorbance in the sample to referencesamples with a known concentration of thrombomodulin (a standard curve).

When the concentration of thrombomodulin in the patient plasma samplehas been determined, this will either be above or below thepredetermined threshold level of at least 2.5 ng/ml, preferably 4 ng/ml.

-   -   Patients with thrombomodulin values above the threshold level        suffer from systemic endotheliopathic syndrome and are        candidates for treatment with prostacyclin and analogues        thereof.    -   Patients with thrombomodulin values below the threshold level do        not have systemic endothelial damage and will not be candidates        for treatment with prostacyclin and analogues thereof.

Preferably, the thrombomodulin test is be a quick point-of-care (POC)test based on whole blood input so a drop of whole blood is placed ontoe.g. a lateral flow assay (stick) or another immunoassay platform likee.g. cartridges based on microfluidics technology, to display a resultwithin 2-5 minutes reporting whether a patient has a thrombomodulinlevel above or below a predetermined threshold level.

Treatment Initiation

Immediately after the test result is available i.e., from minutes toapproximately 2-3 hours after blood sampling pre-hospital or patientadmission/recognition of acute critical illness when using the ELISAassay described above or 15-30 min after blood sampling pre-hospital orpatient admission/recognition of acute critical illness if using thequick whole blood test or POC assay as described above, the patientstarts treatment with an intravenous infusion of prostacyclin oranalogues thereof in a dose of 0.5-4 ng/kg/min, preferably 1-2ng/kg/min.

To administer the treatment through an intravenous infusion system, theprostacyclin or analogues thereof either has to be diluted in anappropriate infusion media (e.g. 0.9% saline) to obtain an appropriateconcentration of prostacyclin and analogues thereof in the infusionfluid or the prostacyclin and analogues thereof are available in aready-to-use appropriate infusion solution.

An appropriate amount of prostacyclin and analogues thereof diluted inthe intravenous infusion fluid corresponds to a 24-30 hour weightadjusted dose in a small volume, e.g., 100-500 ml. For example, in apatient with a body weight of 70 kg treated for 24 hours with 1ng/kg/min prostacyclin and analogues thereof as a 4 ml/hour intravenousinfusion, this corresponds to a concentration of prostacyclin andanalogues thereof of 105 μg/100 ml in the infusion dilution bag (1ng/kg/min*70 kg*60 min*25 hours=105,000 ng/100 ml=105 μg/100 ml).

For each 24 hours, a new infusion dilution of prostacyclin and analoguesthereof will be prepared and administered (as described above) ascontinues intravenous infusion to the patient for the following 24hours.

The treating staff (pre-hospital emergency staff, medical doctors,nurses etc.) has simple instructions for how to prepare an appropriateinfusion dilution of the treatment for a patient with a given weight.The instructions should be flexible so the treating staff can eitheradminister the treatment as a fixed volume infusion (with variableconcentration of prostacyclin and analogues thereof in the infusionfluid, adjusted to patient weight) or a variable volume infusion (with afixed concentration of prostacyclin and analogues thereof in theinfusion fluid), depending on the type of infusion the treating staffnormally use pre-hospital or in their Department/ICU/ward.

During the treatment with prostacyclin and analogues thereof the patientreceives normal standard of care i.e., supportive care e.g. ventilation,fluid therapy, blood transfusion, antibiotics, vasopressor therapy,damage control surgery, specific drugs used for the specific acutecritical illness etc. The therapy is thus an add-on to the normalstandard of care treatment regimen.

Optimally the intravenous infusion with prostacyclin or analoguesthereof in the dose should begin as early as possible after thethrombomodulin test results has become available i.e., within 30 min-12hours after known test results, pre-hospital as well as in-hospital. Thetime to test should not exceed 6 hours after presentation.

Monitoring Treatment Response and Deciding Length of Treatment

The intravenous infusion with prostacyclin and analogues thereof shouldcontinue for a minimum of 72 hours (3 days) up till several weeks.

After the first 3 treatment days, the circulating thrombomodulin levelshould be measured to reveal whether this has decreased, increased orhas remained unchanged compared to the baseline (pre-treatment) value.If the thrombomodulin level has decreased from baseline (e.g. a drop of10% or preferably by 20% or more) and the patient has improvedclinically judged by the attending physician (e.g., reduced need forsupportive care), the intravenous infusion with prostacyclin andanalogues thereof can be ceased so the patient receives just standardcare. If however the thrombomodulin level has increased (>10% increase)or remained unchanged from baseline or the patient has not improvedclinically judged by the attending physician (e.g., increased need forsupportive care), the intravenous infusion with prostacyclin andanalogues should continue for the next 24 hours as an add-on to standardcare.

After another 24 hours treatment (total 4 treatment days), thecirculating thrombomodulin level should be measured to reveal whetherthis has decreased, increased or has remained unchanged compared to thebaseline (pre-treatment) value. If the thrombomodulin level hasdecreased from baseline (by the aforementioned >10% drop or, preferably,by the aforementioned >20% drop) and the patient has improved clinicallyjudged by the attending physician (e.g., reduced need for supportivecare), the intravenous infusion with prostacyclin and analogues thereofcan be ceased. If however the thrombomodulin level has increased (>10%increase) or remained unchanged from baseline or the patient has notimproved clinically judged by the attending physician (e.g., increasedneed for supportive care), the intravenous infusion with prostacyclinand analogues should continue for the next 24 hours as an add-on tostandard care.

This loop of thrombomodulin guided prostacyclin treatment continues forup till several weeks, or until the patient has recovered clinically toan extend that allows discharge from ICU or receives anstop-resuscitation-code or dies.

After the last 24 hours treatment (until a total of treatment time of uptill several weeks), the intravenous infusion with prostacyclin andanalogues thereof is ceased.

During the treatment with prostacyclin and analogues thereof, thepatient receives standard of care.

Optimally, thrombomodulin values should be measured on a daily basis todecide if the treatment should continue for another 24 hours or shouldcease.

Example 1 Evidence of Mechanistic Link

A mechanistic link between systemic endotheliopathic syndrome andmortality in acute critically ill patients was first indicated to theinventors in a study of ˜1,200 septic patients who were followed dailywith blood samples from intensive care unit (ICU) admission and thefollowing four days. Here, the inventors observed that patients who hada reduction in their levels of soluble thrombomodulin (a recognizedcrude marker of endothelial cell damage) over that time had an increasedsurvival rate compared to patients with stable or increasing levels ofthrombomodulin as an indicator of continued endothelial damage.Similarly, the inventors found that patients suffering from severeinfection (from local infection to septic shock), trauma, acutemyocardial infarction or resuscitated cardiac arrest all present withthe same type of systemic endothelial damage, and across all patientsthe inventors found that thrombomodulin is the strongest marker formortality.

Together this supports the new notion relevant for the presentdisclosure that various types of acute critically ill patients maydevelop the same disease, systemic endotheliopathic syndrome, which canbe diagnosed by clinical determination of an acute critical illness andhigh levels of circulating soluble thrombomodulin

Evidence for Medical Use of Prostacyclin and Analogues Thereof forTreatment of Systemic Endotheliopathic Syndrome

The inventors have investigated the anti-thrombotic potential ofprostacyclin with functional whole blood hemostatic assays proven tocorrelate with clinical bleeding conditions and transfusion requirements(thrombelastography (TEG) and impedance aggregometry (Multiplate)) andsurprisingly they discovered that low-dose prostacyclin infusion had nomeasurable anti-thrombotic effects.

The inventors investigated (WO 2013/143548, examples 2 and 3) healthyvolunteers and acute critical illness and found that in healthyvolunteers, 1-4 ng/kg/min prostacyclin infusion neither influenced clotformation nor platelet aggregation. Furthermore, in acute critically illpatients undergoing major abdominal surgery (Example 6) undergoing PCIafter acute myocardial infarction [Holmvang et al 2012, Example 5],0.5-1 ng/kg/min prostacyclin infusion neither influenced clot formationnor platelet aggregation.

Importantly, the inventors surprisingly found that intra- andpost-operative prostacyclin infusion was associated with improved clotformation in patients undergoing major abdominal surgery (Example 6).The finding that low-dose prostacyclin infusion (0.5-1 ng/kg/min) inacute critically ill patients does not compromise hemostasis or inducebleeding or adverse events related to impaired hemostasis is inaccordance with several other studies of acute critically ill patientstreated for hours-to-days with low-dose (0.5-4 ng/kg/min) prostacyclininfusion: Traumatic brain injury patients (0.5-2 ng/kg/min for up to 72hours) [Grande et al 2000; Naredi et al 2001], liver transplantationpatients (4 ng/kg/min for 6 days and 1 ng/kg/min for 7 days,respectively) [Barthel et al 2012; Neumann et al 1999], CABG patients (2ng/kg/min for up to 48 hours) [Morgera et al 2002], patients with septicshock (1 ng/kg/min for 24 h and a dose increasing cardiac index 15% (1-2ng/kg/min) for several hours, respectively) [Kiefer et al 2001; Lehmannet al 2000], patients undergoing surgery for acute lower limp ischemia(0.5-2 ng/kg/min for 6 hours/day for 4-7 days) [de D G et al 2006; de DG et al 2007] and patients undergoing elective open repair of abdominalaortic aneurysm (0.8-1.2 ng/kg/min for 72 h) [Beirne et al 2008].

In conclusion it has now been found that prostacyclin dose-dependentlyinhibits platelet aggregation but low-dose prostacyclin infusion (up to4 ng/kg/min) but does not compromise hemostasis or induce bleeding oradverse events related to impaired hemostasis in healthy volunteers andacute critically ill patients.

Example 2 Reevaluation of a Prior Art Study

Healthy Volunteer Study: Safety and Efficacy of 4 ng/kg/min i. v.Flolan® Infusion in Healthy Subjects, Reported in WO 2013/143548.

The study was conducted investigating the influence of 2 hours i.v.Flolan® (epoprostenol sodium, prostacyclin analog) infusion at a dose of4 ng/kg/min in eight healthy male volunteers.

Endothelial function was evaluated in the study e.g. by measuring plasmalevels of endothelial derived biomarkers by commercially available ELISAkits. Biomarkers indicating: Endothelial glycocalyx damage (syndecan-1),endothelial cell damage (thrombomodulin) or necrosis (nucleosomes,HMGB1), endothelial cell activation (PAI-1) and endothelial cellanticoagulation (protein C, antithrombin) were investigated (FIG. 2).

Prostacyclin in the administered dose did not change blood pressure orheart rate from baseline values at any time point during the studyperiod. Furthermore, no changes were observed in Multiplate and TEGvalues comparing baseline and later values, indicating that 4 ng/kg/mini.v. prostacyclin infusion does not influence hemodynamics orhemostasis.

The administered dose of prostacyclin was observed to have anendothelial protective effect evidenced by a marked decrease in thecirculating level of thrombomodulin, an effect that appeared to beprolonged continuing for several hours after ceasing the prostacyclininfusion. Also, the circulating level of protein C decreased in thehours after ceasing the prostacyclin infusion, indicating thatprostacyclin enhanced activation of protein C. The circulating level ofPAI-1, an inhibitor of fibrinolysis shed from the activated endothelium,also declined further indicating that the prostacyclin infusiondeactivated the endothelium and enhanced endogenous fibrinolysis.Finally, the circulating level of antithrombin decreased indicating thata higher amount of antithrombin was attached to the endothelialglycocalyx rather than being circulating in its soluble form i.e.providing the endothelium with improved anticoagulant properties.

The finding that the administered dose of prostacyclin was associatedwith concurrent decreases in circulating thrombomodulin, protein C,PAI-1 and antithrombin in healthy individuals is reported here as aproof-of-concept of the endothelial protective effect of prostacyclin ofimportance for its capacity to treat systemic endotheliopathic syndrome.

Conclusion:

In this study the inventors found that i.v. infusion of a prostacyclinanalog at a dose applied widely clinically (4 ng/kg/min) neitherinfluenced hemodynamics nor whole blood hemostatic competence. Thefinding that the prostacyclin analog protected the endothelium supportsthe notion, that administration of low-dose prostacyclin or prostacyclinanalogs, in particular iv-administration, is useful for treatment ofsystemic endotheliopathic syndrome.

Example 3 Reevaluation of a Prior Art Study

Healthy Volunteer Study: Efficacy of 1 ng/kg/min i. v. Ilomedin®Infusion in Healthy Subjects. Reported in WO 2013/143548.

A study was reported investigating the influence of 2 hours i.v.Ilomedin® (iloprost, prostacyclin analog) infusion at a dose of 1ng/kg/min in eight healthy male volunteers. Blood samples forendothelial function were collected at the following time-points: Beforethe infusion (0 h), after 30 min infusion (30 min), immediately afterceasing the infusion (2 h) and 2 hours after the end of iloprostinfusion.

Endothelial function was evaluated by measuring plasma levels ofendothelial derived biomarkers by commercially available ELISA kits.Biomarkers indicating: Endothelial glycocalyx damage (syndecan-1),endothelial cell damage (thrombomodulin) and endothelial cellanticoagulation (protein C) were investigated. Furthermore, we measuredplasma concentration of prostacyclin (FIG. 3).

The administered dose of iloprost had an endothelial protective effectevidenced by a marked decrease in the circulating level ofthrombomodulin, an effect that appeared to be prolonged continuing forseveral hours after ceasing the iloprost infusion. Also, the circulatinglevels of protein C and Syndecan-1, the latter a biomarker of glycocalyxdamage, decreased during iloprost infusion, indicating that prostacyclinenhanced activation of protein C (resulting in a decline in thenon-activated form of protein C) and protected the glycocalyx. Finally,the circulating level of prostacyclin increased approximately 15% duringiloprost infusion confirming that systemically detectable increases inprostacyclin were obtained by low-dose 1 ng/kg/min iloprost infusion.

Conclusion:

This study support the notion that an endothelial protective effect oflow-dose i.v. infusion with a prostacyclin analog (1 ng/kg/min,iloprost, ilomedin®) supporting the capacity of administration oflow-dose prostacyclin or prostacyclin analogs, in particulariv-administration, for treatment of systemic endotheliopathic syndrome.

Example 4 Revaluation of Prior Art

Patient Study: Safety and Efficacy of Pre-Filter Flolan® Infusion inCritically Ill Patients [Windelov et al 2010]

Furthermore, in a retrospective study of intensive care patients needingcontinuous renal replacement therapy, the inventors found that patientswho received prostacyclin in the dialysis filter had lower 30-daymortality compared to patients who received heparin in the filter as ananticoagulant (21% vs. 39%)

The inventors conducted a retrospective study of critically ill patientstreated or not treated with prostacyclin infusion during their intensivecare unit (ICU) stay [Windelov et al 2010]. Ninety-four critically illpatients admitted to the ICU for medical or surgical complications,underwent hemofiltration (continuous renal replacement therapy, CRRT)with or without concomitant Flolan® (epoprostenol sodium, prostacyclinanalog) treatment. Flolan® was administered at a low dose (5 ng/kg/min)in the filters to prevent these from clotting and consequently, therewas only a minor spill-over of Flolan® to the systemic circulation.

The study revealed that the two groups (prostacyclin vs heparin) werecomparable with regard to disease severity (APACHE II score) at ICUadmission but during their ICU stay (before CRRT), prostacyclin patientsappeared more severely ill evidenced by more patients having septicshock, disseminated intravascular coagulation (DIC), severethrombocytopenia and a lower platelet count at start of hemofiltration(Table 1).

During CRRT with either prostacyclin or heparin, platelet countincreased in the prostacyclin patients whereas it declined in theheparin patients, indicating reduced disease severity in the patientstreated with prostacyclin (Table 1). Importantly, when comparingmortality, the prostacyclin patients tended to have decreased mortalityat 30 days (21% vs. 39%, p=0.12), 90 days (34% vs. 53%, p=0.10) and 365days (38% vs. 57%, p=0.09).

Conclusion:

The finding of increased platelet count and reduced mortality in CRRTpatients receiving prostacyclin in the filters indicate that the minorspill-over of prostacyclin to the systemic circulating influences theendothelium beneficially in acute critically ill patients by limitingconsumption of platelets, microvascular occlusion, organ failure andmortality, i.e. hallmarks of systemic endotheliopathic syndrome.

Example 5 Revaluation of Prior Art

Patient Study: Safety and Efficacy of 0.5 ng/kg/min i.v. Ilomedin®Infusion in PCI Patients [Holmvang et al 2012]

The inventors conducted a randomized placebo controlled double blindclinical trial investigating safety and efficacy of 24 hours i.v.ilomedin® (iloprost, prostacyclin analog) infusion at a dose of 0.5ng/kg/min compared to placebo in 17 patients with acute myocardialinfarction (AMI) undergoing percutanuous coronary intervention (PCI)with stent implantation [Holmvang et al 2012]. Patients randomized to 24hours active (n=9) or placebo (n=8) therapy were evaluated forhemodynamics, bleeding events and for functional whole blood hemostasis(Multiplate, TEG) and endothelial function in blood sampled beforeiloprost infusion (baseline), during infusion (1 h, 6 h and 24 h) and 24hours after ceasing iloprost infusion (48 h).

Bleeding was evaluated by GUSTO criteria (severe, moderate, mild, none)and hemostasis was evaluated by Multiplate and TEG according to themanufactures recommendations. Endothelial function was evaluated bymeasuring plasma levels of endothelial derived biomarkers bycommercially available ELISA kits. Biomarkers indicating: Endothelialglycocalyx damage (syndecan-1), endothelial cell damage(thrombomodulin), endothelial cell activation (sE-selectin, ICAM-1) andanticoagulation (protein C) were investigated.

The study revealed that patients in the two groups (iloprost vs placebo)were comparable with regard to baseline demography and disease severity.Furthermore, no differences in hemodynamics, bleeding events, Multiplateor TEG were observed between groups during or after iloprost infusion.

Importantly, the inventors found that sE-selectin, a biomarkerreflecting endothelial activation, decreased significantly from baselineto 48 h in the iloprost patients whereas it increased in the placebopatients (FIG. 4) difference between patient groups p=0.008), indicatingan endothelial protective effect of iloprost continuing after ceasingiloprost infusion.

Conclusion:

AMI PCI patients treated with 24 hours i.v. prostacyclin infusiondisplayed evidence of prolonged reduction in endothelial activationcontinuing 24 hours after ceasing the infusion. The prostacyclininfusion did not negatively influence hemodynamics, bleeding events orhemostasis. This study supports the notion that prolonged low-dose i.v.prostacyclin infusion in acute critically ill patients is safe andeffective in protecting the endothelium and hence capable of treatingsystemic endotheliopathic syndrome.

Example 6 Safety and Efficacy of 1 ng/kg/min i.v.

Patient Study: Safety and Efficacy of 1 ng/kg/min i. v. Ilomedin®Infusion in Whipple (Major Abdominal Surgery) Patients

The inventors conducted a randomized placebo controlled double blindclinical trial investigating safety and efficacy of intra- andpost-operative i.v. ilomedin® (iloprost, prostacyclin analog) infusionat a dose of 1 ng/kg/min compared to placebo in 16 patients undergoingwhipple surgery at Rigshospitalet, a tertiary level teaching Universityhospital in Copenhagen, Denmark. The inclusion criteria were adults (age8 years) undergoing whipple surgery based on surgical indication due tocancer/dysplasia, familial adenomatous polyposis orinflammation/pancreatitis. However, all included patients had cancer asa surgical indication: C. pancreas n=12; C. papilla vateri n=2; C.ductus choledochus n=1; Cholangiocarcinoma n=1. The patients wererandomized to receive i.v. infusion with iloprost (1 ng/kg/min, activetreatment, n=8) or saline (0.9%, placebo treatment, n=8) at an equalvolume during surgery (intra-operative: median ˜5 hours) and 6 hoursafter surgery (post-operative), yielding a total active/placebo infusiontime of ˜11 hours.

Data on demography, co-morbidities, surgical time and 28-day mortalitywere assessed. Hemodynamics were measured continuously (heart rate (HR),systolic, diastolic and mean arterial pressure (SBT, DBT, MAP)) or atbaseline, end of surgery and 6 h and 24 h post-operative (systemicvascular resistance (SVR), stroke volume (SV), central venous pressure(CVP)). The volume and number of units of red blood cells (RBC), plasmaand platelet concentrates administered were recorded at the end ofsurgery, 6 h and 24 h post-operative. Bleeding volume was assessedintra-operative and 6 h and 24 h post-operative. The volume ofcrystalloids and colloids (only albumin was allowed) was, together withurine output, recorded intra-operative and 6 h and 24 h post-operative.

Blood samples for evaluation of functional whole blood hemostasis (TEG)and endothelial function were collected pre-commencing infusion withstudy drug (baseline), at the end of surgery (post-op) and 6 h aftersurgery just before ceasing study drug infusion (6 h). Blood samples forroutine biochemistry (hemoglobin, creatinine and lactate) were collectedat baseline, post-op and 6 h and 24 h post-operative.

TEG was performed according to the manufactures recommendations.Endothelial function was evaluated by measuring plasma levels ofendothelial derived biomarkers by commercially available ELISA kits.Biomarkers indicating: Endothelial glycocalyx damage (syndecan-1),endothelial cell damage (thrombomodulin) and necrosis (nucleosomes) wereinvestigated.

Comparing iloprost and placebo patients, no differences were found withregard to demography, comorbidities, surgical indication or 28-daymortality. Furthermore, the groups received a similar volume of fluidsintra- and post-operative and displayed comparable changes inhemodynamics.

However, with regard to transfusion requirements, hemostasis andendothelial function, the inventors surprisingly found major differencesbetween the treatment groups as iloprost patients had reduced RBCtransfusion requirements, reduced increases in lactate, improvedfunctional hemostasis and reduced endothelial damage (FIG. 5 abc).

Thus, in brief, placebo patients received more intra- and post-operativeRBC compared to iloprost patients (median 0 ml (IQR 0-163) vs. 695 ml(IQR 0-969), p=0.083) and a significantly larger volume of RBC fromarrival at the post-operative ward and 6 h post-operatively (iloprostpatients 0 ml (IQR 0-0) vs. placebo patients 115 ml (IQR 0-296),p=0.027). Furthermore, placebo patients tended to have lowerpost-operative hemoglobin compared to iloprost patients (median 6.2mmol/l (IQR 5.9-6.4) vs. 6.7 mmol/l (IQR 6.5-7.7), p=0.058) andpost-operatively lactate increased in placebo patients but remainedunchanged in iloprost patients indicating that iloprost infusionimproved microvascular perfusion. Considering hemostatic competence,placebo patients displayed impaired intra-operative hemostaticcompetence as TEG maximum clot firmness decreased intra-operatively inplacebo patients (p=0.034) whereas it remained stable in iloprostpatients (p=NS).

Importantly, the inventors found that iloprost patients displayedevidence of improved endothelial protection and reduced endothelialdamage compared to placebo patients (FIG. 5 abc) as thrombomodulin andnucleosome levels, markers of endothelial cell damage and necrosis,increased significantly more intra- and postoperatively in placebopatients compared to iloprost patients (FIG. 5 ab). Also, the inventorsobserved a trend towards higher levels of syndecan-1 in placebo patientscompared to iloprost patients (FIG. 5c ) indicating that iloprostprotected the glycocalyx. Together these findings indicate that iloprostinfusion exerted an endothelial protective effect in acute criticallyill surgical patients.

Conclusion:

Whipple patients treated with iloprost infusion intra- andpost-operatively received fewer RBC transfusions, had improvedhemostatic capacity, improved microvascular perfusion and evidence ofreduced endothelial damage together supporting a capacity ofprostacyclin infusion to treat systemic endotheliopathic syndrome.

Example 7 Revaluation of Prior Art

The inventors have measured soluble thrombomodulin levels in bloodplasma from 2,118 acute critically ill patients suffering from trauma,myocardial infarction, cardiac arrest, sepsis, severe sepsis and septicshock (Table 2). The blood sample for thrombomodulin measurement wastaken immediately upon admission to the hospital or immediately afterobservation of the occurrence of the acute critical illness.

The inventors discovered that the acute critically ill patients could bestratified into a group with low mortality rate and a group with highmortality rate based on the measured thrombomodulin level. Thus, a highlevel of thrombomodulin in blood plasma was associated with high 28-daymortality whereas a low thrombomodulin level in blood plasma wasassociated with low 28-day mortality.

Table 2 details a study of median thrombomodulin levels in plasma/serum(ng/ml) in 2,118 acute critically ill patients suffering from trauma,myocardial infarction, resuscitated cardiac arrest, sepsis, severesepsis or septic shock, stratified according to gender and 28-dayoutcome (survival vs. mortality).

The inventors discovered that there was a significant difference inthrombomodulin levels between survivors and non-survivors across alldiseases (p<0.0001), but no difference in thrombomodulin between maleand female (p=0.114).

FIG. 6 displays the receiver operating characteristic (ROC) curve ofthrombomodulin for predicting 28-day mortality in acute critically illpatients suffering from trauma, myocardial infarction, cardiac arrest,sepsis/severe sepsis/septic shock. Soluble thrombomodulin level areaunder the curve (AUC) for predicting 28-day mortality is 0.744(0.712-0.776), p<0.0001, with the highest Youden Index revealing athreshold level of 4.0 ng/ml thrombomodulin in blood plasma. At the 4ng/ml threshold level, sensitivity is 0.781 and 1-specificity is 0.407of the test for prediction of 28-day mortality. Based on these data, 85%of the patients whom die before 28 days have thrombomodulin >4 ng/ml.

Based on the inventors data, choosing a lower threshold level ofthrombomodulin results in increased true-positive rate (sensitivity) andincreased false-positive rate (1-specificity) whereas choosing a higherthreshold level of thrombomodulin results in reduced true-positive rate(sensitivity) and reduced false-positive rate (1-specificity) (Table 3).Consequently, both a lower and higher threshold level of thrombomodulinwill result in a lower Youden Index and hence lower performance of thediagnostic test.

It should be noted that a thrombomodulin level of 4 ng/ml in plasmacannot be expected to be identical to the thrombomodulin level measuredin whole blood since the plasma fraction of whole blood is onlyapproximately 55%. (Discussed below based on data comparing thethrombomodulin level in plasma and whole blood).

Conclusion:

Acute critically ill patients with blood or blood plasma thrombomodulinvalues above 4 ng/ml suffer from systemic endotheliopathic syndrome andwill benefit from treatment with prostacyclin or analogs thereof.

Due, however, to the observed benefits of the present invention; theinventors suggest to operatively use a lower threshold level than 4ng/ml (plasma/serum) as an indicator for treatment with prostacyclin oran analogue thereof according to the present invention. A thresholdlevel corresponding to a plasma/serum level of 2.5 ng/ml is consideredsatisfactory in the method of the invention or values in between 2.5ng/ml to 4 ng/ml, such as e.g. 3.0 or 3.5 ng/ml thrombomodulin inplasma/serum.

Selecting e.g. a threshold level of 2.5 ng/ml will result in an almost91% capture of treatment suited subjects (true positive), however at thecost of a larger number of patients being unnecessarily treated withprostacyclin or an analogue thereof (false positive). Due to the highsafety profile and low risk of adverse effects in treatments using thesuggested low-dose prostacyclin or analogues thereof, it seemsreasonable to proceed in such a manner, in particular since latercontrol according to the invention will limit the unnecessary treatmentexposure received by patients not in need thereof.

Example 8 Trauma

Table 4a details results based on 635 trauma patients. It was found thatthe level of sTM (soluble thrombomodulin) at admission in traumapatients depended on injury severity (increases most in patients withhigh abbreviated injury severity score (AIS) for abdomen and thoraxi.e., severe abdominal and thorax injuries) including shock degreereflecting that the proportion of patients with sTM above 2.5 ng/mldiffer between cohorts of severely (84.9%) and moderately (45.2%)injured patients (Table 4a).

Table 4b details results based on 270 trauma patients. It was found thatin the first 24 h and 72 h, the level of sTM increases in 64% and 80% ofthe moderately injured patients, respectively, and from 24 h to 72 h,sTM increases in 61% of the patients. The absolute and proportionalchange in sTM from admission and 72 h onward is displayed in Table 4b,ranging from a median increase of 0.31-0.72 ng/ml corresponding to12-34% increases.

It was observed that:

The Level of sTM at Baseline and During Follow-Up is Linked toComplications Mortality

When comparing the sTM level in survivors and 28d non-survivors, the sTMlevel at admission (median IQR) (2.95 ng/ml (1.73-4.21) vs. 2.29 ng/ml(1.44-3.38)), at 24 h (3.92 ng/ml (3.7-5.55) vs. 2.94 ng/ml (2.2-4.32))and 72 h (4.56 ng/ml (3.95-5.85) vs. 3.35 ng/ml (2.67-4.6)) is higher innon-survivors compared to survivors (p=0.047, p=0.016 and p=0.076,respectively).

Sepsis

High sTM level at admission tend to be associated with increased risk ofsepsis the first 28d (ROC for admission sTM on development of sepsiswithin 28d is AUC 0.594 (95% Cl 0.487-0.700), p=0.085).

Renal Replacement Therapy (RRT) (Single Organ Failure)

sTM levels at 24 h and 72 h tend to be associated with increased risk ofrenal replacement therapy (RRT) during the first 28d (ROC for 24 h sTMAUC 0.771 (0.556-0.985), p=0.068 and ROC for 72 h sTM AUC 0.877(0.789-0.966), p=0.072)) emphasizing that high sTM levels afteradmission are also linked to organ failure.

The Change in sTM Level from Admission and Onward is Linked toComplications

Bleeding

sTM increases from 24-72 h in more patients who experience bleedingswithin the first 24 h of admission than in patients who do not bleed theinitial 24 h (76% vs. 50% display sTM increases, p=0.063).

Renal Replacement Therapy (RRT) (Single Organ Failure)

sTM increases from 0-24 h, from 0-72 h and from 24-72 h in all (100%)patients who during the first 28 days require RRT due to development ofkidney failure (single organ failure). Furthermore, an increase in sTMthe initial 24 h from admission (0-24 h) is associated with increasedrisk of RRT (ROC for sTM change 0-24 h on RRT within 28 d is 0.794(0.619-0.969), p=0.048). Also, the change in sTM from 0-24 h ispositively correlated with RRT days (kidney failure) (both p<0.05)emphasizing that increases in sTM the first 24h from admission isassociated with enhanced development of organ failure.

Ventilation (Single Organ Failure)

Also, the change in sTM from 0-24 h is positively correlated withventilator days (length of ventilator treatment i.e., respiratoryfailure) emphasizing that increases in sTM the first 24 h from admissionis associated with enhanced development of organ failure.

Disease severity scores for trauma were: Injury Severity Score (ISS)[evaluated at admission], Glasgow Coma Scale (GCS) score [evaluated atadmission], and Sequential Organ Failure Assessment (SOFA) score[evaluated daily in the ICU].

Together, these findings indicate that early and continued measurementof blood or plasma thrombomodulin levels in trauma patients will allowidentification of high-risk patients i.e., patients with systemicendothelial damage and thus patients with the highest thrombomodulinlevels or highest increases in thrombomodulin. High-risk patients withhigh thrombomodulin levels due to systemic endothelial damage willbenefit from therapy with low-dose prostacyclin or analogs thereof,which will be safe even in trauma patients due to the low-dose regimen.

Example 9 Cardiac Arrest

Based on n=169 patients resuscitated from cardiac arrest. It was foundthat the level of sTM at admission in resuscitated cardiac arrestpatients depends on the time in shock and the depth of shock (higher sTMwith longer time from CA to ROSC and with lower pH and higher lactate),the catecholamine level (higher sTM with higher adrenaline doseadministered) and the age of the patient (higher sTM in older patients).

Table 5a details the proportion of OHCA patients with sTM>2.5-5-5 ng/mlat admission, 24 h, 48 h and 72 h. In the first 24 h, 48 h and 72 h, thelevel of sTM increases in 35%, 52% and 43% of the patients compared tobaseline and sTM increases in 77% from 24-48 h, in 38% from 24 h-72 hand in 16% from 48 h-72 h. The median absolute changes from 0-24 h, from0-48 h and from 0-72 h in all patients are 0.27 ng/ml, 1.08 ng/ml and0.80 ng/ml, respectively.

Table 5b details the absolute and proportional (median) changes in sTMfrom admission and 24 h and 72 h onward in 28 day survivors andnon-survivors. Importantly, the change in sTM in 28 d survivors comparedto non-survivors differs significantly, with the most pronounceddifference being that sTM remains unchanged in survivors the first 24 hwhereas it increases 16% in non-survivors.

Table 5c details the predictive value of sTM at different time-pointsfor poor cognitive outcome (CPC≥3/mRS≥4) evaluated by ROC analysis. Ascan be learned from the results continued high sTM levels remain closelylinked to high mortality and poor outcome.

It was observed that:

The Level of sTM at Baseline and During Follow-Up is Linked toComplications Mortality

When comparing the sTM level in 28 d survivors and non-survivors, thesTM levels at admission, 24 h, 48 h and 72 h are all increased innon-survivors compared to survivors: Admission (7.78 ng/ml (6.36-11.41)vs. 6.56 ng/ml (5.17-8.31), p<0.0001), 24 h (9.04 ng/ml (6.28-11.61) vs.6.6 ng/ml (5.28-9.27), p<0.0001), 48 h (9.96 ng/ml (7.3-12.85) vs. 7.79ng/ml (6.31-10.52), p<0.006) and 72 h (8.46 ng/ml (5.95-12.16) vs. 7.23ng/ml (5.46-9.91), p=0.058).

Poor Neurologic Outcome

Also, high sTM level at admission and after 24 h, 48 h and 72 h allpredict poor cognitive outcome evaluated by the CPC and mRS scores(Table 5c).

The Change in sTM Level from Admission and Onward is Linked toComplications

The increase in sTM from admission to 24 h, 48 h and 72 h onward allcorrelate with the time in and depth of shock (time from CA to ROSC, pHand lactate) and the administered adrenaline dose (all p<0.05)emphasizing that the severity of the initial “hit” (shock,catecholamines) drives progressive endothelial damage and henceincreases in sTM.

Mortality

The magnitude of the sTM increase from 0-24 h is associated withmortality evidenced by ROC: Higher sTM increase from 0-24 h isassociated with increased 28 d mortality with AUC 0.587 (0.484-0.689),p=0.103.

Poor Neurologic Outcome

Also, the magnitude of the sTM increase from 0-24 h is associated withcognitive outcome evidenced by ROC: The predictive value of the sTMincrease from 0-24 h is associated with CPC (AIC 0.566 (0.469-0.633),p=0.192) and mRS≥4 (AUC 0.578 (0.480-0.676), p=0.124) indicating thatprogressive increases in sTM are associated with poor cognitive outcome.

Disease severity scores for cardiac arrest were: Cerebral PerformanceCategory (CPC), and modified Rankin Scale (mRS) [cardiac arrest].

Together, these findings indicate that early and continued measurementof blood or plasma thrombomodulin levels in patients resuscitated fromcardiac arrest will allow identification of high-risk patients i.e.,patients with systemic endothelial damage and thus patients with thehighest thrombomodulin levels or highest increases in thrombomodulin.High-risk patients with high thrombomodulin levels due to systemicendothelial damage will benefit from therapy with low-dose prostacyclinor analogs thereof, which will be safe in patients resuscitated fromcardiac arrest due to the low-dose regimen.

Example 10 Myocardial Infarction

Based on n=571 STEMI patients. It was found that the level of sTM atadmission in patients with acute myocardial infarction (ST segmentelevation myocardial infarction, STEMI) is linked to disease severityand outcome with higher sTM levels in patients with more severe disease(highest levels in patients with shock and ICU requirement).

Table 6 details the proportion of OHCA patients with sTM>2.5-5-5 ng/mlat ICU admission (data from two different cohorts of septic patients).

It was observed that:

The Level of sTM at Admission is Linked to Mortality and ComplicationsMortality

When comparing the level of sTM at admission in 28-day and 90-daynon-survivors and survivors, the 28-day (3.23 ng/ml (2.23-4.16) vs. 2.09ng/ml (1.56-2.96), p<0.0001) and 90-day (3.23 ng/ml (2.20-4.16) vs. 2.10ng/ml (1.56-2.96), p<0.0001) non-survivors have higher sTM levelscompared to survivors. sTM at admission correlates inversely with daysto death (p=0.045) reflecting that high sTM is associated with fasterprogression to death. By ROC analyses, admission sTM is a strongpredictor of 28-day (AUC 0.718 (0.635-0.801), p<0.0001) and 90-day (AUC0.713 (0.633-0.792), p<0.0001) all cause mortality and of 28-day (AUC0.736 (0.648-0.824), p<0.0001) and 90-day (AUC 0.726 (0.639-0.812),p<0.0001) cardiovascular disease mortality.

Disease Severity

Admission sTM correlates positively with Troponin I (a biochemicalmarker for myocardial injury) (p=0.008) by ROC analysis sTM isassociated with shock before PCI (AUC 0.580 (0.495-0.666), p=0.061) andwith risk of ICU admission (AUC 0.605 (0.510-0.701), p=0.053). Also, sTMis a strong predictor of 28-day (AUC 0.640 (0.555-0.725), p=0.007) and90-day (AUC 0.644 (0.564-0.724), p<0.0001) congestive heart failure.

Together, these findings indicate that early and continued measurementof blood or plasma thrombomodulin levels in patients with myocardialinfarction will allow identification of high-risk patients i.e.,patients with systemic endothelial damage and thus patients with thehighest thrombomodulin levels or highest increases in thrombomodulin.High-risk patients with high thrombomodulin levels due to systemicendothelial damage will benefit from therapy with low-dose prostacyclinor analogs thereof, which will be safe in myocardial infarction patientsdue to the low-dose regimen. It is expected that prostacyclin therapy inmyocardial infarction patients treated with PCI (percutaneous coronaryintervention) will prevent and treat sequelae from the injurious hitsuch as myocardial infarction with shock, ischemia-reperfusion,catechol-amines etc.

Example 11 Sepsis/Severe Sepsis/Septic Shock

Based on two different cohorts (n=749 and n=184) of patients withsepsis/severe sepsis/septic shock it was observed that the level of sTMat admission in patients with sepsis/severe sepsis/septic shock isclosely linked to disease severity with higher sTM levels in patientswith higher disease severity scores (APACHE II, SOFA) and hence higherdegree of organ dysfunction i.e., higher lactate, lower blood pressure,lower platelet count and hemoglobin, lower urine output, higher BUN andcreatinine.

It was observed that

The Level of sTM at Admission is Linked to Mortality and ComplicationsMortality

When comparing the level of admission sTM in 7-day, 28-day and 90-daynon-survivors and survivors, sTM is increased in the 7-day (9.21 ng/ml(6.74-12.79) vs. 7.35 ng/ml (4.77-10.35), p=0.001), 28-day (9.34 ng/ml(6.86-12.5) vs. 6.81 ng/ml (4.47-9.66), p<0.0001) and 90-day (9.11 ng/ml(6.31-11.92) vs. 6.66 ng/ml (4.61-9.7), p=0.006) non-survivors comparedto survivors.

sTM at admission correlates inversely with days to death (p=0.001)reflecting that high sTM is associated with faster progression to death.By Cox proportional hazards models, the admission sTM level is a strongpredictor of 7-day, 28-day and 90-day mortality (all p<0.001) and by ROCanalysis, sTM is a strong predictor of mortality: 7-day mortality AUC0.65 (0.56-0.75), p=0.005; 28-day mortality AUC 0.68 (0.60-0.76),p<0.001 and 90-day mortality AUC 0.63 (0.54-0.71), p=0.004.

Disease Severity Scores

sTM at admission correlates strongly positively with SAPS II score(p<0.0001) and SOFA score (p=0.003), and for each 1 ng/ml increase insTM, SAPS II score increases 1.32 (95% Cl 0.73-1.91) (p<0.001) pointsand SOFA score increases 0.17 (95% Cl 0.07-0.27) points (p=0.001).

Bleeding and Transfusion Requirements

The sTM level at admission predicts general bleeding in the ICU (ROC AUC0.598 (0.498-0.678), p=0.059), serious bleeding (requiring >3 RBC) (ROCAUC 0.612 (0.506-0.717), p=0.068) and upper GI bleeding (ROC AUC 0.680(0.551-0.809), p=0.037). Also, admission sTM predicts need for RBC (ROCAUC 0.605 (0.517-0.693), p=0.018), FFP (ROC AUC 0.582 (0.495-0.669),p=0.067) and platelets (ROC AUC 0.614 (0.523-0.705), p=0.016) during ICUstay.

Renal Replacement Therapy (RRT) (Single Organ Failure)

Admission sTM predicts the 90-day need for dialysis therapy by ROC withAUC 0.688 (0.595-0.782), p<0.001.

Together, these findings indicate that early and continued measurementof blood or plasma thrombomodulin levels in patients with sepsis, severesepsis, or septic shock will allow identification of high-risk patientsi.e., patients with systemic endothelial damage and thus patients withthe highest thrombomodulin levels or highest increases inthrombomodulin. High-risk patients with high thrombomodulin levels dueto systemic endothelial damage will benefit from therapy with low-doseprostacyclin or analogs thereof, which will be safe in sepsis, severesepsis, or septic shock patients due to the low-dose regimen.

Disease severity scores for sepsis were: Simplified Acute PhysiologyScore II (SAPS II) [evaluated at admission], and Sequential OrganFailure Assessment (SOFA) score [evaluated daily in the ICU] [sepsis].

Together, these findings indicate that early and continued measurementof blood or plasma thrombomodulin levels in patients with sepsis/severesepsis/septic shock will allow identification of high-risk patientsi.e., patients with systemic endothelial damage and thus patients withthe highest thrombomodulin levels or highest increases inthrombomodulin. High-risk patients with high thrombomodulin levels dueto systemic endothelial damage will benefit from therapy with low-doseprostacyclin or analogs thereof, which will be safe in these patientsdue to the low-dose regimen.

Example 12 Rapid Method of Identifying Patients with SystemicEndotheliopathic Syndrome

The inventors have discovered a new disease entity, systemicendotheliopathic syndrome. Patients with systemic endotheliopathicsyndrome suffer from acute critical illness and concurrent evidence ofsystemic endothelial cell damage identified by increases in circulatinglevels of a specific marker reflecting endothelial cell damage.Consequently, identification of patients with systemic endotheliopathicsyndrome among acute critically ill patients can be done by measuringcirculating levels of thrombomodulin, a specific marker of endothelialcell damage with a high sensitivity for diagnosing systemicendotheliopathic syndrome. The fact that patients with systemicendotheliopathic syndrome can be identified by a pre-determinedthreshold level of circulating thrombomodulin introduces personalizedmedicine into the invention, which is opposite to the “one size fitsall” strategy currently used in medical care. Personalized medicinerefers to therapy fitted to the individual patient based on riskstratification, disease phenotype, genetic profile etc.

Accordingly, acute critically ill patients with systemicendotheliopathic syndrome can be rapidly identified (and diagnosed ashaving that syndrome) using a method that involves measuringthrombomodulin level in a biological sample of the acute critically illpatient, and comparing the measured thrombomodulin level to apredetermined threshold.

Preferably, the thrombomodulin test is a quick point-of-care (POC) testbased on whole blood input so a drop of whole blood is placed onto e.g.a lateral flow assay (stick) or another immunoassay platform like e.g.cartridges based on microfluidics technology, to display a result within2-5 minutes reporting whether a patient has a thrombomodulin level aboveor below a predetermined threshold level.

It is suggested that thrombomodulin may be detected using a quick POCtest such as lateral flow assays (sticks) or other immunoassay platformslike e.g. cartridges based on microfluidics technology, as the time toreceive a result would significantly decrease, e.g. assays could beperformed en route aboard an ambulance between a site of an accident anda trauma center or immediately upon hospital admission/recognition ofacute critical illness. Obtaining a result from a POC test withinminutes (instead of hours) would allow rapid allocation to prostacyclintherapy for patients with systemic endotheliopathic syndrome.

As a proof of concept, that soluble thrombomodulin in whole blood is asufficiently sensitive marker over plasma, the present inventorsperformed a measurement of soluble thrombomodulin by ELISA in wholeblood versus plasma.

Measurement of Soluble Thrombomodulin by ELISA in Whole Blood Comparedto Plasma Background

In the enzyme linked immunosorbent assay (ELISA) method, bound antigen(or antibody) is detected by an antibody linked (primarily orsecondarily) to an enzyme whose activity can be determined. The activityof the antibody-linked enzyme serves as a quantitative estimate of theamount of the investigated antigen (or antibody) in the biologicalspecimen.

In thrombomodulin ELISA, patient sample (typically plasma or serum) isadded to ELISA wells pre-coated with an antibody directed againstthrombomodulin. After a washing step, a detection antibody directedagainst (another epitope on) thrombomodulin is added (the detectionantibody may be primarily or secondarily linked to an enzyme whoseactivity can be determined). The amount of enzyme activity in eachsample is mathematically converted to a concentration measure based onthe enzyme activity in samples with known amounts of thrombomodulin(standard curve).

When the thrombomodulin level in acute critically ill patients is usedto guide therapy it is pivotal that the thrombomodulin test can beperformed rapid. One way to speed up testing is to measurethrombomodulin in whole blood instead of plasma/serum as whole bloodrequires no blood sample processing (it takes ˜15-20 min to prepareplasma and ˜30-60 min to prepare serum from a whole blood sample).

Since not all molecules can be easily detected in whole blood (wholeblood may contain substances that can interfere with specificity of thetest), the inventors investigated if thrombomodulin was directlydetectable in whole blood when measured by ELISA. By conducting thebelow described experiments, the inventors surprisingly discovered thatthrombomodulin could be precisely and accurately detected in wholeblood.

Experiments

Whole blood from 10 healthy human volunteers was sampled (two tubes 4 mlEDTA tubes from each subject). From each person, one tube was spun at3000 rpm for 10 min to generate plasma and one tube was left unprocessed(whole blood). Immediately after the spinning (plasma), plasma and wholeblood was pipetted onto the ELISA plate.

From each subject, the following duplicates of plasma/whole blood wasinvestigated for its thrombomodulin content:

-   -   I) Plasma diluted 1:2 with assay buffer (golden standard and        recommended for the ELISA)    -   II) Whole blood undiluted    -   III) Whole blood diluted 1:2 with assay buffer    -   IV) Whole blood diluted 1:4 with assay buffer        Besides using whole blood as sample material, the ELISA was        conducted according to the manufacturer's recommendations.

The hematocrit values from the healthy volunteers was calculated from anexpected normal hemoglobin concentration of 8.3-10.5 mmol/l in male(mean 9.4 mmol/l) and 7.3-9.5 mmol/l in female (mean 8.4 mmol/l)corresponding to hematocrit values of 42% in female and 48% in malevolunteers (meaning that the plasma content in whole blood was 58% infemale and 52% in male).

Results

Table 8 displays the measured thrombomodulin levels in 1:2 dilutedplasma (golden standard) and in different dilutions of whole blood.

In plasma, the mean thrombomodulin concentration was 1.92 (SD 0.12)ng/ml in the 10 healthy volunteers, with a low intra-assay variationbetween duplicates (CV%, corresponding to high precision) of 6.9%.

In undiluted whole blood, the mean concentration was 0.78 (SD 0.06)ng/ml and after correcting for hematocrit (hct) it was 1.38 (SD 0.30)ng/ml, corresponding to a recovery of 71.5% (SD 5.9%) compared toplasma. Notably, the precision of the test in undiluted whole blood wasextremely high with mean CV % between duplicates only being 8.8% (Table8). Also, the linear correlation between plasma and undiluted wholeblood levels of thrombomodulin revealed a strong Pearson product momentcorrelation, r=0.93, yielding a high coefficient of determination,r²=0.87 (FIG. 9a ) (meaning that 87% of the variation in one variablewas statistically explained by variation in the other variable, i.e. anextremely good fit). The thrombomodulin concentration in plasma andundiluted whole blood was significantly different (p=0.002, pairedt-test) with the correction-factor being (1−71.5)/71.5=1.4, emphasizingthat thrombomodulin could be measured precisely in undiluted wholeblood.

In 1:2 diluted whole blood, the mean concentration was 1.23 (SD 0.33)ng/ml and after correcting for hematocrit (hct) it was 2.16 (SD 0.59)ng/ml, corresponding to a recovery of 110.9% (SD 15.8%) compared toplasma. The precision of the test in 1:2 diluted whole blood wasextremely high with mean CV % between duplicates only being 10.3% (Table8). The linear correlation between plasma and undiluted whole bloodlevels of thrombomodulin revealed a strong Pearson product momentcorrelation, r=0.91, yielding a high coefficient of determination,r²=0.83 (FIG. 9b ) (meaning that 83% of the variation in one variablewas statistically explained by variation in the other variable, i.e. anextremely good fit). The thrombomodulin concentration in plasma andundiluted whole blood was comparable (not different, p=0.296, pairedt-test), emphasizing that thrombomodulin could be measured precisely andaccurately in whole blood diluted 1:2.

In 1:4 diluted whole blood, the mean concentration was 1.04 (SD 0.51)ng/ml and after correcting for hematocrit (hct) it was 1.83 (SD 0.90)ng/ml, corresponding to a recovery of 90.19% (SD 33.4%) compared toplasma. The precision of the test in 1:4 diluted whole blood wasacceptable with mean CV% between duplicates being 21.0% (Table 8). Thelinear correlation between plasma and undiluted whole blood levels ofthrombomodulin revealed a strong Pearson product moment correlation,r=0.93, yielding a high coefficient of determination, r²=0.86 (FIG. 9c )(meaning that 86% of the variation in one variable was statisticallyexplained by variation in the other variable, i.e. an extremely goodfit). The thrombomodulin concentration in plasma and undiluted wholeblood was comparable (not different, p=0.762, paired t-test),emphasizing that thrombomodulin could be measured accurately in wholeblood diluted 1:4.

Conclusion

This experiment demonstrated that thrombomodulin was easily detectablein whole blood. This emphasizes that thrombomodulin has biochemicalfeatures that allows thrombomodulin antibodies to detect this moleculealso in whole blood i.e., a significantly more complex biological samplematrix than plasma. The precision of the thrombomodulin measurement inundiluted whole blood was extremely high and 1:2 dilution of the wholeblood retained precision and improved accuracy.

Based on the results of this experiment, the inventors infer thatthrombomodulin can be easily measured in a whole blood based quick assaylike e.g. a dipstick. This may allow rapid treatment stratification ofacute critically ill patients when this is based on their thrombomodulinlevels.

Qualified Diagnosis of Systemic Endotheliopathy by Adding Measurement ofOther Biomarkers i.e. Syndecan-1, Adrenaline and Vascular EndothelialGrowth Factor (VEGF) in Some Patients

The diagnosis of systemic endothelial damage i.e. systemicendotheliopathic syndrome (SES) in acute critically ill patients may insome patients be further qualified besides that obtained by measuringthrombomodulin levels pre-hospital or at hospital admission/recognitionof acute critical illness. Thus, adding a pre-hospital or at hospitaladmission measurement of syndecan-1 (a biomarker reflecting endothelialglycocalyx damage) and/or adrenaline (a endogenous stress biomarkerdirectly drivers endothelial damage) will aid the diagnosis of “systemicendotheliopathic syndrome”.

Patients, in particular trauma patients, with thrombomodulin levels justabove a given cut-off but with concurrent syndecan-1 and adrenalinelevels above their given cut-offs, suffer from severe systemicendotheliopathic syndrome. These patients, with borderline increasedthrombomodulin but high syndecan-1 and adrenaline levels, are thus athigher-than-expected (based on thrombomodulin) risk of diseaseprogression and organ failure and excess mortality, and these patientswill benefit from prostacyclin therapy as described above. Theprostacyclin therapy should be initiated earliest possible and monitoreddaily by thrombomodulin and clinical disease scores, as described above.

Thus, similar to thrombomodulin, the measurement of syndecan-1 andadrenaline as additional biomarkers that can diagnose and qualify theseverity of systemic endotheliopathy i.e. systemic endotheliopathicsyndrome. Preferably, all biomarkers (thrombomodulin, syndecan-1,adrenaline) are measured either individually or simultaneously by aquick POC assay either pre-hospital or immediately upon hospitaladmission/recognition of the acute critical illness. Applying a quickPOC assay that reveal results within a few minutes, enables fastdiagnosis of systemic endotheliopathic syndrome and hereby a fast riskstratification and initiation of prostacyclin therapy. Prompt initiationof prostacyclin therapy will minimize further progression of theendotheliopathy in the early phase of the acute critical illness andwill thus enable fast reversal and treatment of the endotheliopathy.

Evidence from Trauma

In 635 trauma patients, high syndecan-1 and adrenaline levels werestrong and independent predictors of mortality.

FIG. 10 shows a receiver operating characteristic (ROC)-curve ofsyndecan-1 for predicting mortality in trauma patients. Syndecan-1 AUCfor predicting 28-day mortality: AUC 0.599 (0.537-0.661), p=0.001.Highest Youden Index reveals a threshold level of 40 ng/ml syndecan-1 inplasma. Sensitivity 0.569, 1-specificity 0.354.

FIG. 11 shows a receiver operating characteristic (ROC)-curve ofAdrenaline for predicting mortality in trauma patients. Adrenaline AUCfor predicting 28-day mortality: AUC 0.629 (0.567-0.691), p<0.0001.Highest Youden Index reveals a threshold level of 225 pg/ml Adrenalinein plasma. Sensitivity 0.658, 1-specificity 0.435.

When using the above cut-offs for syndecan-1 (40 ng/ml) and adrenaline(225 pg/ml), the following hazards ratios (HR) for 28-day mortality wererevealed:

-   Plasma syndecan-1 above 40 ng/ml: HR 2.16 (95% CI 1.43-3.24), Wald    14, p<0.0001-   Plasma adrenaline above 225 pg/ml: HR 2.47 (95% CI 1.61-3.80),    Wald=17, p<0.0001

The power of these cut-off can be illustrated by the proportion ofpatients dying within 1-28 days when stratified according to syndecan-1or adrenaline level ad admission.

1-Day Mortality:

Patients with syndecan-1 levels>40 ng/ml at admission have a 4-foldhigher <24 h mortality compared to patients with syndecan-1 levels<40ng/ml (10.4% vs. 2.7%, p<0.0001). Patients with adrenaline levels>225pg/ml have 4-fold higher <24 h mortality compared to patients withadrenaline levels<225 pg/ml (8.5% vs. 2.1%, p<0.0001).

Especially for early mortality (<24 h) risk, adding syndecan-1 andadrenaline measurements and their cut-off levels to that ofthrombomodulin, tends to improve the diagnosis of traumaticendotheliopathy and thus risk stratification allowing early treatmentinitiation (syndecan-1 adding significant value compared tothrombomodulin: p=0.153 and adrenaline adding significant value comparedto thrombomodulin: p=0.085). The strength for early mortality predictionemphasizes the need for fast diagnosis of systemic endotheliopathicsyndrome by POC assays allowing a fast initiation of prostacyclintherapy.

28-Day Mortality:

Patients with syndecan-1 levels>40 ng/ml at admission have a 2-foldhigher 28-day mortality compared to patients with syndecan-1 levels<40ng/ml (26% vs. 14%, p<0.0001). Patients with adrenaline levels>225 pg/mlhave more than 2-fold higher 28-day mortality compared to patients withadrenaline levels<225 pg/ml (25% vs. 11%, p<0.0001)

When applying other cut-offs than 40 ng/ml for syndecan-1 and 225 pg/mlfor adrenaline, the sensitivities and 1-specificities for predictingmortality are obtained (data from 635 trauma patients), as shown inTable 9 and 10, respectively.

Finally, in addition to thrombomodulin and/or syndecan-1 and/oradrenaline, measurements of the vascular endothelial growth factor(VEGF) level in patients suffering from acute critical illness candiagnose patients with systemic endotheliopathic syndrome whom haveexcessive loss of vascular integrity i.e., excessive loss of fluids outof the intra vascular compartment. Risk stratification and prostacyclintherapy based on cut-off levels of VEGF measured pre-hospital or atadmission/recognition of acute critical illness represents yet anothertool to personalize care for acute critically ill patients.

Thus, a patient is considered to have systemic endotheliopathic syndrome(also denoted systemic endotheliopathy or simply endotheliopathy) whenthe patient suffers from acute critical illness AND is diagnosed withsystemic endothelial damage.

1.-38. (canceled)
 39. A method of diagnosing for an individual diagnosedwith an acute respiratory failure due to capillary leakage in the lungscaused by sepsis, if said individual is a candidate for combinationtreatment of standard care including ventilator therapy for said acuterespiratory failure in combination with administration of prostacyclinor an analogue thereof, said method comprising: measuring a baselineconcentration of soluble thrombomodulin in blood or plasma of saidindividual; determining if said baseline concentration of solublethrombomodulin in blood or plasma is above a threshold level of at least2.5 ng/ml; and administering the combination treatment of standard carefor said acute respiratory failure in combination with administration ofprostacyclin or an analogue thereof to said individual period if saidbaseline concentration of soluble thrombomodulin in blood or plasma isabove said threshold level of at least 2.5 ng/ml.
 40. A method accordingto claim 39, wherein said threshold level is at least 4 ng/ml.
 41. Amethod according to claim 39, wherein said method is a method ofdiagnosing severe endothelial damage in said individual diagnosed withsaid acute respiratory failure; wherein said individual is diagnosedwith severe endothelial damage in addition to said acute respiratoryfailure, if said baseline concentration is above said threshold level.42. A method according to claim 39, wherein said method is a method ofdiagnosing systemic endotheliopathic syndrome in an individual diagnosedwith said acute respiratory failure; wherein said individual isdiagnosed with systemic endotheliopathic syndrome when said individualis diagnosed with both said acute respiratory failure and severeendothelial damage.
 43. A method of using thrombomodulin as a biologicalmarker in a method for diagnosing for an individual diagnosed with anacute respiratory failure due to capillary leakage in the lungs causedby sepsis, if said individual is a candidate for combination treatmentwith standard care including ventilator therapy for said acuterespiratory failure in combination with administration of prostacyclinor an analogue thereof, said method comprising: measuring a baselineconcentration of soluble thrombomodulin in blood or plasma of saidindividual; determining if said baseline concentration of solublethrombomodulin in blood or plasma is above a threshold level of at least2.5 ng/ml; and administering the combination treatment of standard carefor said acute respiratory failure in combination with administration ofprostacyclin or an analogue thereof to said individual if said baselineconcentration of soluble thrombomodulin in blood or plasma is above saidthreshold level of at least 2.5 ng/ml.
 44. A method according to claim43, wherein said threshold level is at least 4 ng/ml.
 45. A methodaccording to claim 43, wherein said method is a method of diagnosingsevere endothelial damage in said individual diagnosed with said acuterespiratory failure; wherein said individual is diagnosed with severeendothelial damage in addition to said acute respiratory failure, ifsaid baseline concentration is above said threshold level.
 46. A methodaccording to claim 43, wherein said method is a method of diagnosingsystemic endotheliopathic syndrome in an individual diagnosed with saidacute respiratory failure; wherein said individual is diagnosed withsystemic endotheliopathic syndrome when said individual is diagnosedwith both said acute respiratory failure and severe endothelial damage.47. A method according to claim 43, wherein administration ofprostacyclin or an analogue thereof to said individual includesadministering a dose of 0.5-4 ng/kg/min of prostacyclin or an analoguethereof, preferably 1-2 ng/kg/min, to said individual continuously for afirst time period if said baseline concentration of solublethrombomodulin in blood or plasma is above said threshold level of atleast 2.5 ng/ml.
 48. A method of treating an acute respiratory failurein an individual diagnosed with said acute respiratory failure andundergoing standard care including ventilator therapy for said acuterespiratory failure and concurrent increase in a measured thrombomodulinlevel in blood or plasma of said individual, said method comprising: (a)measuring a baseline concentration of soluble thrombomodulin in blood orplasma of said individual; (b) determining if said baselineconcentration of soluble thrombomodulin in blood or plasma is above athreshold level of at least 2.5 ng/ml; (c) administering a dose of 0.5-4ng/kg/min of prostacyclin or an analogue thereof to said individualcontinuously for a first time period if said baseline concentration ofsoluble thrombomodulin in blood or plasma is above said threshold levelof at least 2.5 ng/ml; (d) measuring at the end of said first timeperiod a concentration of soluble thrombomodulin in blood or plasma ofsaid individual; (e) determining if said concentration of solublethrombomodulin is lower by at least a decrease of 10% compared to saidbaseline concentration of thrombomodulin determined prior to initiationof said prostacyclin administration; (f) assessing if a clinicalimprovement of said acute respiratory failure in said individual hasoccurred during said first time period; and (g) if both a concentrationreduction and a clinical improvement is observed, ceasing prostacyclinadministration while continuing standard care for said acute respiratoryfailure; or (h) otherwise continue prostacyclin administration for asecond time period not exceeding said first time period wherein thesteps (d) to (h) are repeated until ceasing prostacyclin administrationto said individual following step (g).
 49. A method according to claim48, wherein said threshold level is at least 4 ng/ml.
 50. A methodaccording to claim 48, wherein said dose is 1-2 ng/kg/min.
 51. A methodaccording to claim 48, wherein said analogue of prostacyclin is eitherIloprost or Flolan.
 52. A method according to claim 48, wherein saidfirst time period is at least 48 hours or at least 72 hours.
 53. Amethod according to claim 48, wherein said second time period is 12hours or 24 hours.
 54. A method according to claim 48, wherein in step(e) said decrease is at least 20%.
 55. A method according to claim 48,wherein said prostacyclin administration is initiated immediately orshortly after a completion of step (b) has determined that said baselineconcentration of thrombomodulin in blood or plasma is above saidthreshold level.
 56. A method according to claim 48, whereinprostacyclin or an analog thereof is administered as according to step(c) prior to steps (a) and (b) having been completed.
 57. A methodaccording to claim 48, wherein said concurrent increase in a measuredthrombomodulin level in blood or plasma of said individual is the resultof severe endothelial damage.
 58. A method according to claim 39,wherein the measurement of thrombomodulin is conducted by apoint-of-care (POC) assay.
 59. A method according to claim 48 whereinthe measurement of thrombomodulin is conducted by a point-of-care (POC)assay.